A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Although the diversity of its fauna and flora has been studied fairly well, little is known of its microbial communities. In this work, we analyzed the Atlantic Forest ecosystem to determine its bacterial biodiversity, using 16S rRNA gene sequencing, and correlated changes in deduced taxonomic profiles with the physicochemical characteristics of the soil. DNAs were purified from soil samples, and the 16S rRNA gene was amplified to construct libraries. Comparison of 754 independent 16S rRNA gene sequences from 10 soil samples collected along a transect in an altitude gradient showed the prevalence of Acidobacteria (63%), followed by Proteobacteria (25.2%), Gemmatimonadetes (1.6%), Actinobacteria (1.2%), Bacteroidetes (1%), Chloroflexi (0.66%), Nitrospira (0.4%), Planctomycetes (0.4%), Firmicutes (0.26%), and OP10 (0.13%). Forty-eight sequences (6.5%) represented unidentified bacteria. The Shannon diversity indices of the samples varied from 4.12 to 3.57, indicating that the soils have a high level of diversity. Statistical analysis showed that the bacterial diversity is influenced by factors such as altitude, Ca 2؉ /Mg 2؉ ratio, and Al 3؉ and phosphorus content, which also affected the diversity within the same lineage. In the samples analyzed, pH had no significant impact on diversity.

Yellow leaf syndrome (YLS) is a recently reported disease of sugarcane, characterized by yellowing of the leaves. Two pathogens: a virus, Sugarcane yellow leaf virus (SCYLV); and a phytoplasma, sugarcane yellows phytoplasma (SCYP), are associated with the disease. The use of tissue culture was investigated as a means to eliminate both SCYLV and SCYP from exotic varieties undergoing quarantine in Mauritius. Of 43 varieties in quarantine, 28 were infected with SCYLV and 19 with SCYP when checked by RT-PCR and nested PCR, respectively. Seventeen varieties were coinfected with both pathogens. Thirty infected varieties were induced to form callus in vitro using leaf rolls as explants. After two subcultures, 19 varieties were successfully regenerated and tested for SCYLV and SCYP. No pathogen could be detected in any regenerated plantlets. All the regenerated plants remained free from both SCYLV and SCYP over a period of 1 year in the glasshouse, confirming that the pathogens had been eliminated by tissue culture.

Using the polymerase chain reaction (PCR), reverse-transcriptase±PCR (RT±PCR) and double-antibody-sandwich enzyme-linked immunosorbent assay (DAS±ELISA), a phytoplasma (sugarcane yellows phytoplasma, ScYP) and a virus (Sugarcane yellow leaf virus, ScYLV) were detected in sugarcane with yellow leaf syndrome (YLS) in Mauritius. Samples were collected from clones undergoing quarantine, in a variety-collection plot and in commercial fields. A 1´25 kb DNA fragment encoding the phytoplasma 16S rRNA was consistently amplified by nested PCR. Of 134 samples with and without symptoms derived from 113 varieties, 111 were infected by either ScYLV or ScYP. The phytoplasma was detected in 63 samples by PCR. Restriction fragment-length polymorphism (RFLP) analysis of the phytoplasma 16S rDNA amplified product indicated that sugarcane yellows phytoplasma group III, which is related to Western X phytoplasma, is present in Mauritius. ScYLV was detected by RT±PCR and ELISA. The virus was more widely distributed than the phytoplasma, and was found in 70 and 100 samples by ELISA and RT±PCR, respectively. There was a significant correlation between the presence of the phytoplasma and YLS symptoms, while such correlation was not significant for ScYLV detected by RT±PCR. ELISA was less sensitive than RT±PCR for detection of ScYLV. Forty-one samples were coinfected with both microorganisms. Eighty-five per cent of the samples displayed symptoms when ScYLV and SCYP coexisted, while 55 and 38% were observed when ScYP or ScYLV, respectively, was present alone. The results indicate that the presence of both organisms enhanced the syndrome.

To study the phylogenetics of sugarcane (Saccharum officinarum L.) and its relatives we sequenced four loci on cytoplasmic genomes (two chloroplast and two mitochondrial) and analyzed mitochondrial RFLPs generated using probes for COXI, COXII, COXIII, Cob, 18S+5S, 26S, ATPase 6, ATPase 9, and ATPase α (D'Hont et al. 1993). Approximately 650 bp of DNA in the intergenic spacer region between rbcL and atpB and approximately 150 bp from the chloroplast 16S rDNA through the intergenic spacer region tRNA(val) gene were sequenced. In the mitochondrial genome, part of the 18S rRNA gene and approximately 150 bp from the 18S gene 3' end, through an intergenic spacer region, to the 5S rRNA gene were sequenced. No polymorphisms were observed between maize, sorghum, and 'Saccharum complex' members for the mitochondrial 18S internal region or for the intergenic tRNA(val) chloroplast locus. Two polymorphisms (insertion-deletion events, indels) were observed within the 18S-5S mitochondrial locus, which separated the accessions into three groups: one containing all of the Erianthus, Eccoilopus, Imperata, Sorghum, and 1 Miscanthus species; a second containing Saccharum species, Narenga porphyrocoma, Sclerostachya fusca, and 1 presumably hybrid Miscanthus sp. from New Guinea; and a third containing maize. Eighteen accessions were sequenced for the intergenic region between rbcL and atpB, which was the most polymorphic of the regions studied and contained 52 site mutations and 52 indels, across all taxa. Within the Saccharum complex, at most 7 site mutations and 16 indels were informative. The maternal lineage of Erianthus/Eccoilopus was nearly as divergent from the remaining Saccharum complex members as it was from sorghum, in agreement with a previous study. Sequences from the rbcL-atpB spacer were aligned with GENBANK sequences for wheat, rice, barley, and maize, which were used as outgroups in phylogenetic analyses. To determine whether limited intra-complex variability was caused by under sampling of taxa, we used seven restriction enzymes to digest the PCR-amplified rbcL-atpB spacer of an additional 36 accessions within the Saccharum complex. This analysis revealed ten restriction sites (none informative) and eight length variants (four informative). The small amount of variation present in the organellar DNAs of this polyploid complex suggests that either the complex is very young or that rates of evolution between the Saccharum complex and outgroup taxa are different. Other phylogenetic information will be required to resolve systematic relationships within the complex. Finally, no variation was observed in commercial sugarcane varieties, implying a world-wide cytoplasmic monoculture for this crop.