Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): an Invasive species in Central Punjab Pakistan

Physids belong to Class Gastropoda; belong to Phylum Mollusca and being bioindicators, intermediate hosts of parasites and pests hold a key position in the ecosystem. There are three species of Genus Physa i.e. P. fontinalis, Physa acuta and P. gyrina water bodies of Central Punjab and were characterized on the basis of molecular markers High level of genetic diversity was revealed by polymorphic RAPD, however SSR markers were not amplified. The multivariate analysis revealed polymorphism ranging from 9.09 percent to 50 percent among the three Physid species. Total number of 79 loci were observed for the three species under study and 24 loci were observed to be polymorphic. These RAPD fragment(s) can be developed into co dominant markers (SCAR) by cloning and can be further sequenced for the development of the Physa species specific markers to identify the introduced and native species in Pakistan.

Development of SCAR Primers for PCR Assay to Detect Diplodia seriata

The aim of this study was to develop primer pairs for Diplodia seriata identification, one of the most common fungal species associated with grapevine decline in Castilla y León (Spain). Genetic variability of selected isolates of D. seriata was estimated. A molecular marker was generated from a random amplified polymorphic DNA (RAPD) fragment. PCR products of around 1200 bp were obtained with OPE20 primer. The PCR products were cloned and sequenced. The sequences were compared and a fragment of 1207 bp was used to design primer pairs. Two primer pairs were selected (DS3.8 S3-DS3.8 R6 and DS3.8 S3-DS3.8 R4) that amplified a single DNA product of 634 bp and 233 bp, respectively, with D. seriata isolates. No amplification was obtained for any of the 57 isolates of other species. The designed SCAR primer pairs allowed a rapid detection of D. seriata, and were able to detect 0.1 pg of the target DNA. Detection was specific and sensitive for D. seriata. The established protocols detected these fungi in naturally infected grapevines after DNA purification. Diplodia seriata was detectable without DNA purification and isolation in 62.5% to 75% of reactions. The detection of this pathogen in wood samples has great potential for use in pathogen-free certification schemes.

GENETIC, COLORATION, AND GROWTH PERFORMANCE OF TWO DIFFERENT VARIETIES OF Kappaphycus alvarezii

Two different colors (green and brown) of Kappaphycus alvarezii have been farmed in Indonesian waters for many years. This study aimed at comparing two ‘varieties’, i.e. green and brown, both genetically and morphologically. Samples for DNA analysis were collected from a farmer in Pinrang Regency, South Sulawesi. Five universal primers i.e. Ca-01, Ca-02, P-40, P-50, and DALRP were selected to obtain DNA genetic markers in differentiating the green and brown varieties. To compare coloration patterns during cultivation and the growth performance of both varieties, a field experiment was performed in a seaweed farming area in Pinrang Regency, during dry season of August-September 2004. The result of genetic assessment showed that the five selected primers revealed different RAPD banding pattern for both varieties. P-50 and DALRP primers demonstrated the greatest amplification in differentiating RAPD fragment between green and brown varieties. Fragment 900 bp and 1.300 bp were consistently generated in the green variety but were not amplified in the brown variety. The result of the field study confirmed that the coloration pattern of green and brown varieties was fixed; no interchange in color occurred during one crop cultivation.

Genetic Analysis of Inonotus Obliquus Strains by RAPD

Genetic Analysis ofInonotus ObliquusStrains by RAPDRAPD profiling of eightInonotus obliquusstrains isolated from sclerotia collected from different areas of China was conducted to determine the genetic variability within this important medicinal fungus and to better define relationships between the genotype and geographical origins of isolation. Twelve 10-mer primers generated a total of 167 stable and reproducible DNA fragments, of which 101 (60.5%) were polymorphic. DNA fingerprints revealed genetic diversity among the strains tested, but there was the little intraspecific difference between the fingerprints of individual strains. A phenogram constructed based on UPGMA analysis of genetic distances calculated from RAPD fragment data identified three distinct groupings: (1) BCX01 and BCX02, (2) JL01, JL02, JL03, JL04 and JL05, (3) HLJ01. Our data confirm that the genetic variability among different strains may be a useful ancillary tool for identifyingl. obliquussclerotia of different geographical origins.

A PCR Assay Based on a Sequence-Characterized Amplified Region Marker for Detection of Emetic Bacillus cereus

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.

A PCR-based Assay for Detection of Alternaria radicina on Carrot Seed

A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.

Genetic and Morphological Characterization ofCladobotryum Species Causing Cobweb Disease of Mushrooms

ABSTRACT Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.