Title : DNA sequence of geographical diversity of fungal endophytes: Azadirachta indica (Neem)
Abstract:
Endophytes play a significant role in promoting plant growth and enhance plants’ resistance to diseases, pests, and environmental stresses. Host plants provide an environment with sufficient water and nutrients for endophyte establishment. Endophytes have received much attention over the past few decades, yet many questions remain unanswered regarding patterns in their biodiversity at local to global scales. Our research Aim was to categorize the diversity of fungal endophytes in leaves of two plants of medicinal and cultural importance to the indigenous/traditional peoples of Kenya: Azadiracta indica (neem) and Melia azedarach (melea). We then compared this with the endophytes in Arizona USA. Field surveys for endophytes of neem and melea were completed in Kenya and Arizona .Fungal Isolation: Epiphytes were eliminated and surface-sterilized leaf were aseptically cultured on PDA or MEA, incubated in an incubator at 280C for 5 to 7 days. Pure cultures were then used for morphological characterization and Molecular characterization . At the University of Arizona, DNA was extracted from fungal mycelia of the pure cultures .PCR was used to amplify the barcode locus, the internal transcribed spacer of the ribosomal repeat (ITSrDNA). Gel electrophoresis and PCR cleanup was done to prepare samples for Sanger sequencing, which was bidirectional and generated ca. 1000-1200 basepair fragments including both ITSrDNA and a portion of the LSUrDNA, relevant for phylogenetic analysis. Over 175 new endophytes were collected in the study year which brings the total of characterized endophytes to a total of nearly 400 isolates. The endophytes obtained from Melia differ geographically, with distinctiveness between the best-studied region in Kenya (Machakos) and the other regions in Kenya, and between Kenya and Arizona. In Kenya, Phomopsis appears to be a common endophyte of melea, but this was not detected in samples from Arizona. Arizona samples were rich in Rhizopus, Alternaria, and Pyronema, which were not present in the Kenya samples. From neem we obtained a diversity of fungi representing two phyla (Mucoromycota and Ascomycota) and at least five classes, 17 genera, and 20 species from the first 40 isolates for which high-quality DNA sequences were obtained. The endophytes obtained from neem differ geographically as reported for melea as would be expected based on geographic locations of sites. In Kenya, Colletotrichum appears to be a common endophyte of neem, but this was not detected in samples from Arizona. Arizona samples were rich in highly pigmented strains, primarily Dothideomycetes.
