Random Amplified Polymorphic DNA Amplification Research Articles

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

Introduction: For the rapid and accurate genetic identification and authentication of living organisms, improved random amplified polymorphic DNA (RAPD) fragment based development of sequence-characterized amplified region (SCAR) markers is an important genetic technique. Objective: This study aimed to develop SCAR markers for perennial herb Eclipta prostrate (E. prostrate). Methods: Here the RAPD fragments by improved RAPD amplification with primers A11 and N-7 for E. prostrate were cloned into pGEX-T vector, and PCR amplification identified the positive clones. After the enzymatic digestion, they were sequenced with Sanger sequencing. Results: Two SCAR markers were developed, which were very specific to E. prostrate, not found in Penthorum chinense Pursh (P. chinense). The nucleotide sequence search by BLAST GenBank database showed that they are novel in E. prostrate, therefore they were deposited in Genbank with accession number KX671034, KX671035. The markers did not show any identity to other species. Conclusions: Thus, in this study two specific SCAR markers were developed for genetically distinguishing and identifying the plant species E. prostrate from herb P. chinense and others.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

Mei Z, Khan MA, Zhang X, Fu J. 2017. Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers. Biodiversitas 18: 1243-1249. For the genetic identification and authentication of living organisms, development of Sequence-Characterized Amplified Region (SCAR) markers from Random Amplified Polymorphic DNA (RAPD) fragments is a valuable molecular approach. By using SCAR markers, molecular analysis is simplified to a Polymerase Chain Reaction (PCR) analysis using PCR primers designed from specific sequences of the RAPD amplicons. In this study, RAPD fragments from improved RAPD amplification of a perennial herb Penthorum chinense Pursh from China were cloned into a T-vector, and positive clones were identified by PCR amplification, and sequenced with the Sanger sequencing method for the SCAR marker development. Five SCAR markers were developed that were very specific to P. chinense, and deposited in GenBank (accession numbers: KX671029, KX671030, KX671031, KX671032 and KX671033). BLAST searches of these five nucleotide sequences in the GenBank database showed no identity with markers from other species. This study was developing five specific SCAR markers, has enabled reliable genetic identification of the herbal plant species Penthorum chinense Pursh, useful for authenticating future samples of this important medicinal herb.

In vitro Propagation of Crassocephalum crepidioides—An Endangered African Traditional Leaf Vegetable and Molecular Analysis of Micropropagated Plants

ABSTRACTCrassocephalum crepidioides (Benth) S. Moore is an important African traditional leaf vegetable, but its production is very low and its existence threatened. An efficient and reproducible micropropagation protocol of C. crepidioides was developed from nodal stem segments of pot-grown plants. Shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with benzineamino purine (BAP) alone and in combination with zeatin and naphthalene acetic acid (NAA). Elongated shoots successfully rooted on zeatin alone and in combination with indole butyric acid (IBA). Molecular stability of micropropagated plants was evaluated using random amplified polymorphic DNA (RAPD) markers. Survival of nodal segments in micropropagation medium was greater than 70% in all treatments. The presence of plant growth regulators (PGRs) in MS medium influenced shoot formation, rooting, and number of shoots and roots per explant. The best shoot formation frequency (88.7%), number of shoots per explant (3.7), and shoot length (30 mm) were from a medium containing MS supplemented with 10 mg·L−1 BAP, 15 mg·L−1 NAA, and 1.5 mg·L−1 zeatin. Frequency of root formation and survival of regenerated plants were greater on medium containing half-strength MS salt than medium fortified with full-strength MS salt. The best frequency of root formation (average 87.4%) and survival of regenerated plants (average 95.4%) were from media containing 6.0 mg·L−1 zeatin and 1.0–1.5 mg·L−1 IBA and half-strength MS salt. Micropropagated shoots with well-developed roots were acclimatized under greenhouse conditions in pots containing rich sandy loam soil (pH = 7.2). The RAPD amplification products were monomorphic in micropropagated plants and similar to those of the mother plant. No polymorphism was detected, indicating genetic uniformity of micropropagated plants. This is the first report of micropropagation of C. crepidioides, which will facilitate in vitro mass propagation, conservation, and germplasm exchange of this endangered African vegetable.

Development and significance of SCAR marker QG12-5 for Canarium album (Lour.) Raeusch by molecular cloning from improved RAPD amplification

Sequence-characterized amplified region (SCAR) is a valuable molecular marker for the genetic identification of any species. This marker is mainly derived from molecular cloning of random amplified polymorphic DNA (RAPD). We have previously reported the use of an improved RAPD technique for the genetic characterization of different samples of Canarium album (Lour.) Raeusch (C. album). In this study, DNA fragments were amplified using improved RAPD amplified from different samples of C. album. The amplified DNA fragment was excised, purified from an agarose gel and cloned into a pGM-T vector; subsequently, a positive clone, called QG12-5 was identified by PCR amplification and enzymatic digestion and sequenced by Sanger di-deoxy sequencing method. This clone was revealed consisting of 510 nucleotides of C. album. The SCAR marker QG12-5 was developed using specifically designed PCR primers and optimized PCR conditions. This SCAR marker expressed seven continuous "TATG" [(TATG)n] tandem repeats, which was found to characterize C. album. Subsequently, this novel SCAR marker was deposited in GenBank with accession No. KT359568. Therefore, we successfully developed a C. album-specific SCAR marker for the identification and authentication of different C. album species in this study.

Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst.

Development of sequence-characterized amplified region (SCAR) markers from random-amplified polymorphic DNA (RAPD) fragments is a valuable molecular approach for the genetic identification of different species. By using SCAR markers, molecular analysis is reduced to a simple polymerase chain reaction (PCR) analysis using primers designed from the amplicon sequence of RAPD. In this study, the DNA fragments from an improved RAPD amplification of Ganoderma species were cloned into a pGM-T vector; positive clones were identified by PCR amplification and enzymatic digestion, and finally, DNA fragments were sequenced using the Sanger sequencing method for developing the SCAR markers. Two SCAR markers, named LZ4-1 with 534 nucleotides, and LZ5-2 with 337 nucleotides were identified, which are specific to Ganoderma lucidium (Leysser: Fr) Karst species. BLAST of these two nucleotide sequences in the GenBank database showed no identity to other species. We deposited these sequences into the GenBank database (LZ4-1 accession No. KM391933, LZ5-2 accession No. KM391934). PCR assays confirmed them as novel molecular markers for G. lucidium (Leysser: Fr) Karst, which might be used for genetic authentication of adulterant samples. Thus, our study developed two specific SCAR markers for identifying and distinguishing the medicinal mushroom G. lucidium (Leysser: Fr) Karst from other Ganoderma species.

Application of the Random Amplified Polymorphic DNA (RAPD) Fingerprinting to Analyze Genetic Variation in Community Associated-Methicillin Resistant Staphylococcus Aureus (CA-MRSA) Isolates in Iran.

The aim of this study was to apply RAPD technique to analyze the genetic variability among the Iranian CA-MRSA isolates.The RAPD amplification was implemented on 25 strains isolated from the anterior nares of 410 healthy children using four randomly selected oligonucleotide primers from the stocks available in our laboratory, including the primers 1254, GE6, OLP6 and OLP13 from our stock. The amplified PCR products were detected on a 1.5% agarose gel and subjected to further analysis to establish the band profiles and genetic relationships using the Gel Compar® program.The Iranian CA-MRSA isolates produced distinct RAPD patterns which varied based on the primer used, however, the primer 1254 revealed highly polymorphic patterns consisting 5 discernable RAPD types (RT), “RT1” (12, 48%), “RT2” (8, 32%), “RT3” (3, 12%), and “RT4 and RT5”, (a single RAPD type each, 4%). Phylogenetic analysis based on RAPD profiles divided most of the CA-MRSA isolates into 2 distinct but related RAPD clusters, a small group and two single unrelated RAPD types.This study shows that the simple and cost-effective but rather difficult to optimize RAPD fingerprinting could be used to evaluate genetic and epidemiological relationships of CA-MRSA isolates on condition that the patterns are obtained from carefully optimized laboratory tests.

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn. by improved RAPD amplification and molecular cloning

article i nfo Background: Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results: The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions: This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.

English

Genetic diversity within Gerbera jamesonii Bolus is the key to genetic improvement of this important ornamental species. In the present study, genetic diversity of 12 accessions of gerbera was assessed through random amplified polymorphic DNA (RAPD) markers. A total of 40 RAPD primers belonging to OPD, OPE, OPF and OPG series of universal primers set were used out of which, only 10 primers produced clear, reproducible and scorable bands. Ten (10) decamer RAPD primers produced a total of 49 scorable bands from 12 genotypes of G. jamesonii Bolus, out of which 42 were polymorphic and seven were monomorphic. The percentage of polymorphism ranged from a maximum of 100.00% by OPE-02, OPE-14, OPF-18, OPG-18, OPG-16 and OPG-17 to a minimum of 50.00% by OPE-08. The RAPD profiles generated were further evaluated for studying the Jaccard’s similarity coefficient. The average genetic similarity coefficient for the 12 accessions evaluated by Jaccard index was 0.66 ranging from 0.35 to 0.86. The RAPD amplification data were used to obtain similarity matrix and for generation of dendrogram using unweighted pair group method with arithmetic averages (UPGMA) method. Based on the dendrogram, all 12 genotypes could be distinctly divided into two clusters and the accession CF Orange was found most dissimilar from other accessions. This study shows that DNA based molecular marker RAPD is a powerful, less time consuming and cost effective molecular technique for assessment of genetic diversity among different genotypes of G. jamesonii Bolus. The availability of these gerbera RAPD markers would facilitate the use of molecular markers in gerbera breeding and genetic studies.   Key words: Gerbera jamesonii Bolus, molecular markers, genetic diversity, RAPD.

Genetic characterization and authentication of Lonicera japonica Thunb. by using improved RAPD analysis

In traditional medicine, Lonicera japonica (Thunb.) has a notable place, and it has been used for thousands of years in China, Japan, Korea and other East-Asian countries for treating cancer, inflammation, hepatic complications, influenza and wounds. However, the molecular or genetic characteristic of this plant is not well defined. In this study, improved random amplified polymorphic DNA (RAPD) has been employed for the genetic characterization of five varieties of L. japonica collected from different geographic locations of Southern China. A total of 147 bands of DNA fragments were obtained in RAPD-PCR by using 18 primers, and the band sizes ranged from approximately 300-2,000 bp, with 3-11 amplified bands for each primer. Based on the RAPD amplification profiles, cluster dendrogram was obtained, which showed that the similarity coefficients among five varieties of L. japonica ranged from 0.59 to 0.77. To our knowledge, this is the first report of genetic characterization of L. japonica using improved RAPD analysis which has been validated by ISSR analysis, and this characterization may be useful for the preservation of genetic diversity and Lonicera population identification. Moreover, as an option, the improved method could be employed for a variety of applications in genetic diversity and fingerprinting analyses.

Unusual “Flesh-Eating” Strains of Escherichia coli

Monomicrobial necrotizing fasciitis (type II) is typically caused by group A streptococcus alone or in combination with Staphylococcus aureus. Escherichia coli has been isolated from polymicrobial or Fournier's gangrene but has rarely been reported in monomicrobial necrotizing fasciitis. We describe the clinical characteristics and outcomes of seven cases of monomicrobial E. coli necrotizing fasciitis and/or severe soft tissue infection diagnosed at a single institution during an 18-month period. Four isolates from three patients and two isolates from two patients with type I polymicrobial severe soft tissue infection (controls) were assayed by the randomly amplified polymorphic DNA (RAPD) analysis for fingerprinting and PCR amplification of primers in order to detect cytotoxic necrotizing factor 1 and 2 (cnf1 and cnf2) genes. All patients had some type of immune suppression. The limb was the most commonly involved organ. In all cases, E. coli was isolated as a monomicrobial pathogen from blood, fascia, or both. All patients died during hospitalization, three within the first 48 h. The RAPD amplification assay showed a high degree of genetic diversity among the "flesh-eating" strains and controls. The cnf1 toxin gene was identified in two out of three cases, but not in the controls. cnf2 was not detected in any of the patients. E. coli may be responsible for life-threatening necrotizing fasciitis. Further research is needed to reveal relevant risk factors, reservoirs, and modes of transmission of cnf1 E. coli.

Identification of superior varieties of tea (Camellia sinensis (l.) o. kuntze) in the selected UPASI germplasm using biomarkers

Biomarkers are used as a vital tool in cultivar improvement programme for woody perennial tree crops such as (Camellia sinensis (L.) O. Kuntze). Commercially important fifteen accessions were selected and investigated for total polyphenol oxidase (PPO) activity (U/mg of protein), based on the PPO activity range, the accessions were further separated into three groups, namely high (P/11/10, UPASI-16, UPASI-18, UPASI-14, I/30/17), moderate (UPASI-3, UPASI-17, UPASI-13, UPASI-1, UPASI-21) and low (I/30/9, p/11/15, I/30/30,MGL-16, MGL- 8) PPO activity. To study the pattern of genetic diversity, random amplified polymorphic DNA (RAPD) analysis was performed using twenty decamer primers. The RAPD amplification results revealed that genetic similarity (GS) among the accessions tested ranged 0.64 to 0.918 with an average of 0.28%. RAPD dendogram showed three distinct clusters of high, moderate and low using un-weighed pair-group method for arithmetic averages analysis (UPGMA) method. This genetic diversity studies on tea showed effectively for the initial assessment of partitioning the intra specific level of genetic variation correlated to the total PPO enzyme activity. Key words: Camellia sinensis, random amplified polymorphic DNA (RAPD), polyphenol oxidase, genetic diversity.

SOMACLONAL VARIATION AMONG SOMATIC-EMBRYO DERIVED PLANTS OF OLEA EUROPAEA L “CV. KRONEIKI”

Frequency of somaclonal variation in embryogenic calli and plants of Olea europaea L cv.Kroneiki derived from somatic embryogenesis were examined by randomly amplified polymorphic DNA (RAPD) analysis. Radicle and cotyledon (proximal part) of mature zygotic embryo were cultured in OMc medium supplemented with IBA (5 mgL-1) and 2-ip (0.5 mgL-1). Somatic embryogenesis was induced in calli of radicles (55%) and proximal part of cotyledons (8%) on OM medium without any hormones. After culturing the embryogenic calli on OM medium in the presence of BAP (2 mgL-1), regenerated plants were obtained. DNA sample from the leave of the mother plants, seedlings (germinated from zygotic embryo), plantlets (regenerated from somatic embryo), embryogenic calli, non-embryogenic calli and zygotic embryos were subjected to RAPD analysis. 20 arbitrary decamer primers produced polymorphic amplification products. These primers gave a total of 315 reproducible fragments from which 221 (71%) were useful as polymorphic bands with an average of twelve RAPD marks obtained for each primer. A dandogram, based on the unweighted pair group mean average (UPGMA) method of cluster analysis, were constructed using a similarity matrix derived from the RAPD amplification generated by all primers. The estimation of genetic similarity coefficient based on RAPD band sharing data indicated that regenerated plants were less than 75% similar to mother plants.

Genetic variation and polymorphism in the Himalayan nettle plant Urtica dioica based on RAPD marker

Urtica dioica is a medicinal plant and is distributed throughout the ranges of Himachal Pradesh. The Genetic variation was found in U. dioica plant samples, with respect to plant distribution in middle and lower Himalayas. Genetic variability was based on change in altitude. Examination of random amplified polymorphic DNA (RAPD) markers from six plant samples collected at different heights from sea level indicated that genetic variation was appreciable, as samples from lower altitudes showed low genetic similarity with samples collected from higher altitudes. Eight Random decamer primers were used for RAPD amplification. The RAPD products ranged from 500 - 2800 bp. A total number of 134 RAPD markers were obtained from 8 primers, out of which 27 were polymorphic (20.2%) and the rest were monoporphic (79.8%). The polymorphism ranged from 4 - 50%.

Assessment of Genetic Variation Within Indian Mustard (Brassica juncea) Germplasm Using Random Amplified Polymorphic DNA Markers

Genetic diversity among 45 Indian mustard (Brassica juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession 'Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.

Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root‐knot nematode (Meloidogyne spp.)

A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp. DNA was extracted from individual second‐stage juvenile (J2) nematodes of Meloidogyne spp. and amplified by PCR (polymerase chain reaction). Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA. This enabled a molecular diagnostic key for M. incognita, M. javanica, M. arenaria, M. mayaguensis, M. hapla, M. chitwoodi and M. fallax to be designed. The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically. The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species. The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described.

RAPD markers for discriminating tea germplasms at the inter-specific level in China

For the discrimination of tea germplasms at the inter-specific level, four tea species and varieties (Camellia sinensis, C. sinensis var. assamica, C. sinensis var. pubilimba, C. sinensis var. kucha) and their 20 wild allied species (C. sp.) preserved in the China National Germplasm Tea Repositories (CNGTR) were investigated using randomly amplified polymorphic DNA (RAPD) markers. Fifteen primers were chosen from the 61 screened for RAPD amplification. The average DNA polymorphic frequency of RAPD primers at the inter-specific level was 0.30, varying from 0.16 to 0.60, lower than that at the intra-specific level. Using the presence, sometimes absence of unique RAPD markers, it was possible to discriminate 14 of the germplasms investigated. No single primer could discriminate all the 24 germplasms. However, OPO-13 provided rich band patterns and it could discriminate 10 genotypes. The combination of two and three primers made it possible to discriminate 15 and 21 germplasms, respectively. Furthermore, the combination of band patterns or the DNA fingerprinting based on specific RAPD markers generated by OPO-13, OPO-18, OPG-12 and OPA-13 allowed the discrimination of all 24 germplasms investigated. Therefore, RAPD markers also provide a powerful tool to differentiate tea germplasms at the inter-specific level.

Suitability of RAPD for analyzing spined and spineless variant regenerants of pineapple (Ananas comosus L., Merr.)

A random amplified polymorphic DNA (RAPD) analysis of spineless (variant phenotype) plants obtained from micropropagated dormant pineapple (Ananas comosus L., Merr.) axillary buds was performed using arbitrary 10-mer oligonucleotide primers. This was done to investigate the genetic fidelity of the regenerants and to distinguish these variants from regenerants bearing the normal spined phenotype. Of the 58 arbitrary primers used, 29 produced bands unique to the spineless phenotype, and 30 produced bands unique to the spined phenotype. A total of 914 bands were scored, 55 of which were polymorphic to the spineless phenotype and 51 of which were polymorphic to the spined phenotype. On the basis of RAPD amplification products, genetic similarity was estimated in both types of regenerants using similarity coefficients (Nei and Li, 1979). The characteristic finger-prints generated by each probe emphasize genetic variability of regenerants. This technique is suitable for analyzing variant regenerants induced in vitro.

Exposure to Xenobiotics Deeply Affects the Bacteriocenosis in the Rhizosphere of Helophytes

Upon exposure to the aromatic xenobiotics 2,6-dimethylphenol and 4-nitrophenol, distinct changes occurred in the microbial structure of the rhizosphere of the helophytes Carex graclis, Phalaris arundinacea and Juncus effusus (total number, diversity, abundance). Generally speaking, exposure to xenobiotics resulted in an increase in the total numbers of xenobiotic-degrading bacteria on roots and sand particles. The number of bacteria per cm 2 was significantly higher on the root surface than on the surface of sand particles in the rhizosphere. PCR fingerprinting methods such as RAPD (Randomly Amplified Polymorphic DNA) and 16S-23S rDNA intergenic spacer amplification or alternatively rRNA-targeted gene probes for the α-, β-, γ-subclasses of Proteobacteria, the Xanthomonas branch of Proteobacteria and Actinobacteria, were used to determine the diversity of arbitrary samples of bacterial isolates. The diversity of bacteria isolated from the rhizosphere decreased depending on the exposure time. The degrading bacteria were dominated by GRAM-positives. In the case of exposure to 2,6-dimethylphenol of Carex gracilis, GRAM-positive isolates became more dominant as the exposure time increased. Thus it was that after more than six months exposure to 2,6-dimethylphenol, only the GRAM-positives were isolated from a sand-bed reactor planted with Carex gracilis. Some of these isolates show interesting properties in addition to a high 2,6-dimethylphenol degradation rate. The 2,6-dimethylphenol degrading Mycobacterium sp. DMP 20 represents the first fluorescing GRAM-positive bacterium so far described. The introduction of allochthonous, high-performance degraders resulted in Mycobacterium sp. DMP 20 being permanently established in the rhizosphere, whereas the naphthalene degrader Pseudomonas putida PpG7 did not remain in the system.

DNA Fingerprints of Three Different Forms of the Red Palm Weevil Collected from Egyptian Date Palm Orchards

Three morphologically different forms of the red palm weevil (Rhynchophorus ferrugineus Oliv.) were collected from infected date palm fields. The morphological variations detected among different forms included the spotting patterns of black spots on thorax. DNA fingerprinting based on randomly amplified polymorphic DNA (RAPD) was performed. Nine random primers, which gave sufficient amplification products with weevil genome as well as reproducible patterns and sufficient polymophisms, were preselected. Analysis of generated PCR amplification products indicated clearly that three different RAPD patterns peculiar for the three different forms of weevil were produced. These characteristic fingerprints were reproducible for each primer. Two red palm weevil forms, given the numbers 2 and 3, share the majority of RAPD amplification products, meanwhile the form number 1 seems to be genetically far from the two other forms. It could be concluded that the major genetic variations detected herein among the three forms of the red palm weevil may be either due to the generation of new mutant from the weevil or the three forms may be belong to different new varieties. Although the obtained genetic variations are reproducible and stable, even when different random primers were used, it is still early to confirm any of the hypotheses mentioned above and further studies are needed.