Isolation and characterization of hypovirulence associated mycovirus from Fusarium species isolated from apple orchards of Himachal Pradesh

Abstract

newline ABSTRACT newlineFusarium, a genus of filamentous fungi, has many species which serving as important pathogens newlineto many diseases in crops. In general to date, there have not been effective and environmental newlinefriendliness control methods for the fungus. Therefore, utilizing a biological control method is a newlinetactful choice. Hypovirulent or strains infected by mycovirus could be developed as biocontrol newlineagents. An attempt was made to isolate dsRNA elements from Fusarium sp. associated with root newlinerot disease of apple from different apple growing areas of Himachal Pradesh. Mulberry twigs newlineused as baits, were implanted in infected soil of apple orchards for a period of two months. From newlinebaits, Fusarium was isolated and purified. Isolates showing slow growth phenotype were newlineselected and characterized by amplification of ITS and TEF-and#945; regions followed by DNA newlinesequencing of amplified products, which confirmed the identity of fungal isolates as Fusarium newlinesp. Genetic diversity was analysed among Fusarium isolates using Random Amplified newlinePolymorphic DNA. The UPGMA study characterized the fungal isolates on the basis of newlinesimilarity. Cultures which showed slow and abnormal growth phenotype were suspected to be newlineinfected by mycovirus. When inoculated on apple, guava and pear isolate containing newlinehypovirulent factor produced mild symptoms. Presence of mycoviral dsRNA was first confirmed newlineby isolation of dsRNA elements using One Step RNA isolation method and CF-11 cellulose newlinechromatography method specifically for dsRNA isolation followed by RT-PCR amplification newlinewith primer pairs designed from the coat protein sequences of different Fusarium mycoviruses. newlinedsRNA was sent for sequencing and results revealed the presence of virus like sequences which newlinecorresponds to Fowlpox virus (ssRNA), Reticuloendotheliosis virus (ssRNA) and Tomato leaf newlinecurl virus (ssDNA). In addition sequences corresponding to active transposable elements and newlinetransposase were also found. Curing for virus using cycloheximide treatment was done as a newlinepreliminary test. After curing, cultures exhibited normal morphology, pigmentation and normal newlinegrowth rate. The pathogenicity of cured and uncured strains of isolated Fusarium was tested on newlinedifferent fruits. Fusarium strain cured for mycovirus was found to be more pathogenic as newlinecompared to uncured strain. The growth and pathogenicity phenotype was not fully restored in newlinecomparison to wild type. It might be due to presence of transposable elements that interfered newlinewith the revival of the isolate. It indicated that the virus act as hypovirulent factor and inhibited newlinethe pathogenicity of fungus to rot the fruit. newlineKeywords: Mycovirus, ssRNA virus, ssDNA virus, transposable elements, RT-PCR, Curing.