MOLECULAR DETECTION AND IDENTIFICATION OF SOME MICROBES ASSOCIATED TO RED PALM WEEVIL

Abstract

Date palm (Phoenix dactylifera L.) family; Arecaceae, (Angiosperms, monocotyledon) is one of the most important and oldest trees in the arid and semi-arid regions, in which it was cultivated for its edible sweet fruits. It is considered as an economic crop in the Arab land, and Egypt is the first county in date palm cultivation and dates production in the world. This economic tree faces many problems, the biggest one is the Red Palm Weevil (RPW) which known of its rapid expansion and destruction during the last three decades. The knowledge of the red palm weevil gut microbiota composition is of importance, as it could lead to establish new and innovative approaches for red palm weevil management or at least it could help in improving classic management approaches. This study was applied as a first exploration of the gut bacterial community of healthy field caught larvae of Red Palm Weevil (Rhynchophorus ferrugineus), in Egypt, by using culture-independent techniques based on 16S RNA genes. 1. Sample collection Three groups of 20-22 healthy field caught Rhynchophorus ferrugineus larvae – dominated by the late instars – were collected from three different date palm trees from two different places; El-Badrashein and Agriculture Research Center (ARC) in El-Giza governorate, Egypt. The sampling palm trees have a known history clear of any insecticidal or biological control treatment. 2. Larvae dissection and metagenomic DNA extraction Field caught Larvae transferred within 24 hours to the laboratory and undergone dissection and gut content evacuation under aseptic conditions. After dissection process, we have got three pools of larvae gut content in three 50ml tube. Metagenomic DNA extracted from the three gut content pools by using DNA isolation kit (MO-BIO Laboratories).

3. PCR amplification of 16S rRNA genes Bacterial 16S rRNA genes of the bulk DNA of the gut content pools were amplified by using Touch-up and Touch-Down PCR programs, with 341F-GC as a forward primer and 907R as a reverse primer. All of the three pools were amplified positively and produced a 16s rRNA amplicons with an approximate size 586 bp 4. Denaturing Gradient Gel Electrophoreses (DGGE) of 16S rRNA amplicons 16S rRNA amplicons were run on 6% polyacrylamide gel in DCodeTM Mutation Detection System (Bio-Rad,UK). The electrophoresis was conducted at 120 V and 35 Amp under a constant temperature of 60C for 17 hours followed by staining with ethidium bromide for 20 min, washed in double distilled water for 15 min , visualized and photographed under UV gel documentation system; Gel DocTM XR+ imager (Bio-Rad Laboratories inc,UK) 5. DGGE Bands profiling analysis DGGE bands pattern showed 26 distinguishable bands with an average of 8–9 bands per sample. 9 bands were excised, eluted, and undergone Re-PCR amplification with the same primers (without the GC clamp) 6. Sequencing and construction of phylogenetic tree The Bands that shown PCR positive results were purified and sequenced. The sequences were analyzed by nucleotide-nucleotide BLAST, Sequence alignments and Phylogenetic trees were constructed by bioinformatics programs. 7. Results of DGGE profiling and its sequence analysis DGGE bands pattern showed relatively low complexity of the gut microbiota. DGGE bands sequences analysis have shown the relatively prevalence of Lactic Acid Bacteria; Leuconostoc lactis, Lactococcus