Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum.     

Characterization of Kenyan Isolates of Fusarium udum from Pigeonpea [Cajanus cajan (L.) Millsp.] by Cultural Characteristics, Aggressiveness and AFLP Analysis

Isolates of Fusarium udum from pigeonpea (Cajanus cajan) plants with wilt symptoms were collected from various districts in Kenya and were characterized using cultural characteristics, aggressiveness and amplified fragment length polymorphism (AFLP). The 56 isolates of F. udum showed a high level of variability in aerial mycelia growth, pigmentation and radial mycelia growth (colony diameter) on potato dextrose agar. The aggressiveness of 17 isolates of F. udum on seven pigeonpea varieties varied and five aggressive groups were observed in the present study. There were no relationships among cultural characteristics and aggressiveness. AFLP analysis of the 56 isolates was tested for genetic variability using seven primer combinations. A total of 326 fragments was generated of which 121 were polymorphic. Ten AFLP groups were identified among the Kenyan isolates and, although they were not genetically distinct, six AFLP subgroups were genetically distinct. AFLP had no relationship with cultural characteristics, aggressiveness and geographical origin of the isolates. This is the first report on the study of genetic variability of F. udum using DNA analysis.     

Isolation of Three Xylanase-Producing Strains of Actinomycetes and Their Identification Using Molecular Methods

Eighty-eight actinomycetes were isolated from 20 samples collected from different locations in and around Delhi, India. Among these, 69 isolates were found positive for xylanase production. Of 69 isolates, 3 (SN32, SN77, and SN83) produced >125 IU/ml xylanase. Modern genetic tools were used for revealing the identities of these potent xylanase producers. The selected isolates were categorized under the genus Streptomyces based on their cultural and morphologic characteristics. Genetic diversity among these species of Streptomyces was established based on restriction length fragment polymorphism and random amplified polymorphic DNA analysis. The closest phylogenetic neighbours according to the 16S rRNA gene-sequence data for the three isolates SN32, SN77, and SN83 were Streptomyces cyaneus, S. tendae, and S. caelestis, respectively.     

RAPD Analysis of Indian Isolates of Rice Sheath Blight Fungus Rhizoctonia solani

Rice sheath blight fungus Rhizoctonia solani has a wide host range and is highly variable in pathogenecity, sclerotial production and cultural characteristics. In India, breeding for sheath blight resistant cultivars has been a priority area of research. However, lack of adequate information about the genetic variability of the fungal populations occurring in India, non-availability of appropriate markers and the non-availability of resistant donors are some of the limiting factors to achieve this objective. To assess the genetic variability in sheath blight fungus, 18 isolates collected from different rice growing regions of India were analyzed by using random amplified polymorphic DNA (RAPD) markers.The similarity values of RAPD profiles ranged from 0.41 to 0.85 with an average of 0.66 among all the isolates. The percentage polymorphism detected per primer varied from 79.2 to 100%. All the primers could be used to fingerprint the individual isolates. The cluster analysis using unweighted paired group method with arithmetic averages could distinguish between R. solani isolates as well as the virulent and avirulent isolates on rice.     

Genetic variability among isolates and sexual offspring of the plant pathogenic fungus Calonectria morganii on the basis of random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)

Thirty-two strains of the phytopathogenic mold Cylindrocladium scoparium (perfect state Calonectria morganii) isolated from ericaceous hosts and two specimens from the ATCC were examined by random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Five oligonucleotides were chosen as primers for differentiation of the isolates. RAPD patterns of the ATCC strains differed significantly from those of the field isolates. Diversity among field isolates was low. Results obtained in RFLP analysis, with telomere repeats of Neurospora crassa as a probe, were highly consistent with the RAPD data. Isolates were paired in all possible combinations; fertile perithecia occurred in only one combination, from which ascospores were analyzed by formal genetics and RAPD. A bipolar mechanism of homogenic incompatibility was found. Ascospore-derived strains were much more variable than field isolates. Phylogenetic trees suggested a correlation to the host plants from which the strains were isolated. Received: 7 March 1996 / Accepted: 11 April 1996     

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Cultural, morphological, pathogenic and molecular variability amongst tomato isolates of Alternaria solani in India

Early blight (Alternaria solani) is an important disease causing severe damage in tomato. The eleven isolates of A. solani designated as So, Dh, Sh, Va-5, Ka, Ma, Hy, Ba-1, My, Va-3 and Mi were collected from different agroclimatic conditions and these isolates were characterized for cultural, morphological, pathogenic and molecular variations. The pigmentation varied from yellow, brown, black, brownish to greenish black in isolates of A. solani on potato dextrose agar medium. In general, radial growth of all isolates ranged between 14.9 mm and 32.2 mm on PDA and 24.3 mm to 53.7 mm on three selective media i.e., ASM, V-8 juice agar and V-8 juice agar (synthetic) on the fourth day. The fastest radial growth was recorded in the So isolate and slowest in the Ka isolate on PDA, while isolates Dh, Ba-1 and Va-3 were recorded to be faster in growth on ASM, V-8 juice agar and V-8 juice agar (synthetic) medium. The thickness of conidiogenous hyphae varied between 1.17 μ and 9.56 μ, with maximum in the Va-5 and Ma isolates. Most of the isolates showed smooth mycelial growth with circular and irregular margin and without concentric zonation. Sporulation was not found in any of the isolates on four different nutrient media, whereas conidiogenous hyphal length was observed in V-8 juice agar medium only. Based on the pathogenicity, isolates of A. solani were rated as virulent or less virulent based on percentage disease incidence data. Molecular variability studies were also done to find out the best annealing temperature and eighty-six primers were screened to select for maximum polymorphism of DNA. The best annealing temperature was recorded between 32.5 °C and 34.0 °C for the pathogen, and most efficient amplification and polymorphism of DNA was found with random primer 5′-CGCGTTCCTG-3′.     

Molecular characterization and mating type analysis of oyster mushroom (<Emphasis Type="Italic">Pleurotus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>) using single basidiospores for strain improvement

Existence of variability in morphological traits and growth rate of mycelium of homokaryotic single basidiospores can be exploited for the development of inter-strainal hybrids. We isolated 182 single basidiospores from mushroom bodies of P. sajor-caju, P. florida, P. eous and one wild relative of Pleurotus called Hypsizygus ulmaris. The single spores were isolated using a new technique that is less prone to contamination and more efficient than the common techniques used by earlier workers. All the isolates showed a varied range of cultural morphology. Mating types of all the isolates within the species were identified on the basis of hyphal fusion via anastomosis with the tester strains. Four compatible pairs of isolates with well prominent tuft in the contact zone were selected for dikaryon isolation. Dikaryons were used for spawn preparation and mushroom cultivation. The dikaryotic isolates with their replicates were evaluated for spawn run period, yield and biological efficiency. 42 isolates (10 di- and 32 mono-karyotic isolates) were analyzed with RAPD genetic markers. Phenotypic characters and mating types of all the 42 isolates analyzed genetically were correlated with their genetic polymorphism data. The isolates showed very large diversity both at the phenotypic and the genotypic level. Available phenotypic and genotypic data can further help in the selection of monosporous isolates for developing inter-strainal hybrids which can lead to better prospects for genetic improvement in different species of Pleurotus.     

Characterization and correlation of pathogenicity of Botryodiplodia theobromae isolates,the causal agent of black root rot of mulberry (Morus spp.)

Abstract

The study reports the characterization of 10 isolates of mulberry black root rot causing fungus, Botryodiplodia theobromae obtained from the infected gardens of Karnataka, Andhra Pradesh and Tamil Nadu. The analysis based on cultural, morphological, pathogenicity and molecular markers (RAPD and SSRs) revealed significant variations among the isolates. Based on the disease reaction on susceptible V-1 variety, isolates were grouped as pathogenic (60%), moderate pathogenic (20%) and non-pathogenic (20%). Among all isolates, RAPDs revealed higher marker polymorphism however, based on Shannon’s Information Index (I) SSRs were more informative (0.781) compared to the former (0.444). Stepwise Multiple Regression Analysis (MRA) indicated a total of 5, 5 and 3 molecular markers were found to correlate with disease symptoms. Screening of germplasm using multiple strains of virulent isolates will enhance possibilities of locating diverse resistant genes. Pyramiding of these genes will aid in development of mulberry variety with durable resistance and sustainable sericulture.     

Morphological, Pathological and Molecular Variability in Colletotrichum capsici, the Cause of Fruit Rot of Chillies in the Subtropical Region of North-western India

Fruit rot of chillies (Capsicum annuum L.), caused by Colletotrichum capsici under tropical and subtropical conditions, results in qualitative and quantitative yield losses. Based on variation in cultural and morphological traits of C. capsici populations, 37 isolates were categorized into five groups designated, respectively, as Cc‐I, Cc‐II, Cc‐III, Cc‐IV and Cc‐V. In culture, most of the isolates produced cottony, fluffy or suppressed colonies. However, no significant differences were noticed in shape and size of conidia. The reaction of the 37 isolates on an indigenously developed differential set of Capsicum cultivars indicated the existence of different virulences in Himachal Pradesh (HP) chilli populations. Fifteen pathotypes of the pathogen were characterized from various chilli‐growing regions of HP. Pathotype CCP‐1 was most virulent and attacked all the differential cultivars. The genetic relationship between five morphological groups recognized within C. capsici was investigated using random amplified polymorphic DNA (RAPD) analysis. Molecular polymorphism generated by RAPD confirmed the variation in virulences of C. capsici and different isolates were grouped into five clusters. However, four isolates (Cc‐5, Cc‐33, Cc‐29 and Cc‐37) exhibited identical RAPD haplotypes. The pathological and RAPD grouping of isolates suggested no correlation among the test isolates.     

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.     

Isolation and characterization of EST‐derived microsatellite loci from the fungal wheat pathogen Phaeosphaeria nodorum

Eleven polymorphic microsatellite loci and one minisatellite locus originating from expressed sequence tag (EST) libraries of Phaeosphaeria (syn. Stagonospora) nodorum were isolated and characterized. The satellite markers were used to genotype isolates from field populations collected in China, North America and South Africa. The number of alleles per locus ranged from two to 15. Genotype diversity ranged from 87.5 to 95.3 and gene diversity from 0.1 to 0.8. The variable levels of polymorphism within and among populations of P. nodorum renders these 12 satellite loci ideal markers for population genetic analysis of P. nodorum.     

Identification of the New Pathogen (Stemphylium lycopersici) Causing Leaf Spot on Pepino (Solanum muricatum)

Pepino (Solanum muricatum var. pepino) plants were found affected by an extensive leaf spot caused by plant pathogenic fungi during a survey in the Cameron highlands, Pahang state, Malaysia. Symptomatic leaf samples were collected from infected pepino plants and cultivated on PDA medium, and the pathogen was isolated and purified; then, consequently, all isolates were identified as Stemphylium lycopersici on the basis of their cultural and morphological characteristics and combined sequences of the internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) regions. A pathogenicity assay on detached leaves further confirmed that S. lycopersici causes leaf spot disease. To the best of our knowledge, this is the first report of S. lycopersici causing leaf spot on pepino in Malaysia and worldwide.     

Yeasts present during spontaneous fermentation of Lake Erie Chardonnay,Pinot Gris and Riesling

The composition of wine yeast populations, present during spontaneous fermentation of Chardonnay, Pinot Gris and Riesling from the Lake Erie Region was studied. A combination of biochemical and molecular techniques was used to identify non-Saccharomyces and Saccharomyces yeast isolates. The biochemical techniques included analysis of yeast isolates by sugar fermentation and carbon and nitrogen assimilation. Molecular techniques involved ribotyping of a highly variable segment in the 26S rRNA gene using DNA sequence analysis and restriction fragment length polymorphism of amplified DNA. The results show that of the non-Saccharomyces yeasts, several related species of Hanseniaspora, were the most abundant yeasts present during early stages of fermentation. Later in fermentation S. cerevisiae dominated, and based on biochemical analyses consisted of a heterogeneous group of genotypes. There were no major differences in yeast populations among the three types of juice analyzed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular diversity of native Trichoderma isolates against Fusarium oxysporum f. sp. lycopersici (Sacc.). A casual agent of Fusarium wilt in tomato (Lycopersicon esculentum Mill.)

Fusarium wilt in tomato caused by Fusarium oxysporum is the one of the problematic diseases. In this study, 12 native Trichoderma isolates were isolated from different land use types in Rayalaseema region of Andhrapradesh, India and were tested for antagonistic activity against F. oxysporum using dual culture method; the maximum inhibition occurred in WT₂ (78.4%) compared to the control. Molecular characterisation using random amplified polymorphic DNA (RAPD) technique reported 91.8% polymorphism among 12 isolates of Trichoderma. Internal transcribed spacer (ITS) region of rDNA amplification with genus-specific ITS1 and ITS4 universal primers produced amplicon size from 569 bp in all the isolates. The study resulted in identification of good competitive Trichoderma isolates against F. oxysporum. A relationship was found between the polymorphism showed by the Trichoderma isolates and their hardness to F. oxysporum during antagonism. Also, exhibition of sufficient genetic polymorphism aids further exploitation in genomic fingerprinting.     

SSADH Variation in Primates: Intra- and Interspecific Data on a Gene with a Potential Role in Human Cognitive Functions

In the present study we focus on the nucleotide and the inferred amino acid variation occurring in humans and other primate species for mitochondrial NAD⁺-dependent succinic semialdehyde dehydrogenase, a gene recently supposed to contribute to cognitive performance in humans. We determined 2527 bp of coding, intronic, and flanking sequences from chimpanzee, bonobo, gorilla, orangutan, gibbon, and macaque. We also resequenced the entire coding sequence on 39 independent chromosomes from Italian families. Four variable coding sites were genotyped in additional populations from Europe, Africa, and Asia. A test for constancy of the nonsynonymous vs. synonymous rates of nucleotide changes revealed that primates are characterized by largely variable d_N/d_S ratios. On a background of strong conservation, probably controlled by selective constraints, the lineage leading to humans showed a ratio increased to 0.42. Human polymorphic levels fall in the range reported for other genes, with a pattern of frequency and haplotype structure strongly suggestive of nonneutrality. The comparison with the primate sequences allowed inferring the ancestral state at all variable positions, suggesting that the c.538(C) allele and the associated functional variant is indeed a derived state that is proceeding to fixation. The unexpected pattern of human polymorphism compared to interspecific findings outlines the possibility of a recent positive selection on some variants relevant to new cognitive capabilities unique to humans. [Reviewing Editor: Dr. Martin Kreitman] Paola Blasi and Francesca Palmerio contributed equally to the work.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Identification and subtyping of Francisella by pyrosequencing and signature matching of 16S rDNA fragments

Aims: To analyse the V1 region of the 16S rDNA gene by a universal pyrosequencing protocol to identify and subtype Francisella in 31 strains from a repository collection and 96 patient isolates. Methods and Results: Pyrosequencing was used to determine the nucleotide sequence of PCR amplification products of the variable region (V1) of the 16S rDNA from 31 repository strains and 96 isolates from Swedish patients with ulceroglandular tularaemia. Pyrosequencing resulted in a 37 nucleotide sequence, specific for Francisella sp., for all repository strains and patient samples analysed. In addition, the isolates could be divided into two groups based on the analysis of a single nucleotide polymorphism in the sequence: one group included Francisella tularensis ssp. tularensis, ssp. holarctica and ssp. mediasiatica, whereas the other group included Francisella tularensis ssp. novicida and other species of Francisella. The analysis of samples taken from patients suffering from ulceroglandular tularaemia revealed that all isolates belonged to the first group comprising subspecies of F. tularensis virulent for humans. Conclusions: The pyrosequencing analysis of the 16S rDNA V1 is a useful molecular tool for the rapid identification of suspected isolates of Francisella sp. in clinical or environmental samples. Significance and Impact of the Study: Virulent F. tularensis ssp. causing ulceroglandular tularaemia, or those with a potential to be used in a bioterrorism event, could rapidly be discriminated from subspecies less virulent for humans.     

Megaplasmids in Thermus oshimai isolates from two widely separated geographical areas: restriction fragment profiling and DNA homology

Megaplasmid DNA was detected in ten isolates belonging to the recently described thermophilic eubacterial species Thermus oshimai and isolated from hot springs in Portugal (eight isolates) and Iceland (two isolates). The estimated size of the large plasmids purified from T. oshimai SPS-18 from S. Pedro do Sul, Portugal, and from isolate JK-91 from Hveragerdhi-Hengill, Iceland, was 214 and 275 kb, respectively. No sequence homologous to isolate SPS-18 megaplasmid is present in chromosomal DNA as indicated by Southern hybridization analysis. Overall examination of the HindIII fragment profiles of megaplasmid DNAs purified from isolates from the same geographical area gave similar but not always identical restriction profiles on agarose gels. Restriction fragment length polymorphism (RFLP) was higher for megaplasmids present in isolates purified from the Portuguese and Icelandic isolates than for megaplasmids from the same hot spring. Megaplasmid RFLP correlated with previous results obtained on the polymorphism of macrorestriction patterns of whole genomic DNA and with the RFLP of co-resident small plasmid DNA that was found in one half of the isolates examined. The 16-kb HindIII–HindIII fragment from isolate SPS-18 megaplasmid showed DNA–DNA homology with restriction fragments of similar size generated by the large plasmids present in all the other isolates, even in those from hot springs of widely separated geographical areas. This suggests a high degree of sequence conservation in T. oshimai megaplasmids. Received: 12 May 1997 / Accepted: 17 July 1997