Isolation Of Endophytic Fungi From Mangifera Indica Linn And Development Of A Molecular Mothod For Rapid Detection Of Fusarium Mangiferae

Mango malformation disease (MMD), caused by Fusarium, is one of the major diseases affecting mango. This disease is difficult to diagnose and can not be controlled effectively, therefore it presents direct threats to mango production. In China, this disease currently occurs principally in the late-maturing’s mango producing areas, such as Sichuan、Yunnan, and Hot and Dry Valley Basin of Jinsha River. Because the disease takes substantial amount of time to show symptoms, finding a rapid and effective detection technique can provide a theoretical basis to prevent the spread of the disease, control the epidemic, and establish early diagnosing technology. This study analyzed the mango endophytic fungi species and established a rapid detection technique for Fusarium mangiferae. The main works are summarized as follows:Isolation, identification and diversity analysis of endophytic fungi in Mangifera indica Linn:Endophytic fungi isolated from four kinds of Mango were identified and classified based on morphological observations and ITS sequencing analysis. The results were analyzed by such indexes as isolation rate (IR), colonization rate (CR), relative frequency (RF), Shannon-Wiener biodiversity index (H’) and similarity coefficient (Cs). Fifty three strains of endophytic fungi were isolated from480fresh mango specimens. They were identified and classified in to16categories. Among them14belonged to Ascomycota and2to Basidiomycota. The dominant groups of those endophytic strains were Colletotrichum gloeosporioides、Guignardia mangiferae and Botryosphaeria dothidea, accounting for20.75%、18.87%and18.87%respectively. The composition of endophytic fungal community has considerable diversity and difference, and the distribution of endophytic fungi has species and tissue specificity. Based on these results, Fusarium associated with mango malformation has not been isolated from mango yet. The preliminary assessment is that the endogenous fungus of mango does not interfere with the rapid detection.An effective detection technique of mango malformation disease of Fusarium mangiferae:This study was based on ISSR molecular marker. It screened a purpose primer (UBC888) from50primers, and achieved a stable and specific bands at479bp (GenBank No.KJ526382). According to the specific sequence, we designed a pair of SCAR primers (W342, W1772) and got a specific gene fragment at1376bp (GenBank No.KJ526383), which successfully transformed ISSR to SCAR molecular marker. An effective detection technique of mango malformation disease was established based on this pair of primers:The reaction volume of amplification was25μL:the10x PCR buffer (TaKaRa)2.5μL,10mmol/L dNTPs2μL, template (10ng/μL)0.5μL, mixed primers (20μmol/L) lμL, Taq enzyme (5U/μL) (TaKaRa)0.13μL, add ddH20to25μL. PCR conditions:94℃5min;94℃lmin,50℃30s,72℃lmin,45cycles;72℃5min. The technique which we established is of simple、high specificity and sensitivity to the disease both in-vitro and in-vivo, capable of detecting lOpg of fungal DNA. This provided theoretical basis and technical methods for the early diagnosis and proper prevention of mango malformation disease.