Antiviral potential of crude extracts from two Streptomyces spp. against Cucumber Mosaic Virus infection under greenhouse conditions

Marine actinomycetes are a promising source for developing new antiviral agents for plant diseases, as they produce a wide variety of bioactive compounds. To date, antiviral activity of marine actinomycetes against plant viruses are rare. The present study proceeded to identify and characterize promising antiviral actinomycetes from Red Sea in Hurghada, Egypt, control Cucumber Mosaic Virus (CMV) and, profile the individual chemical components of bioactive crude extracts. Biological and molecular characterization was performed to identify CMV isolate using RT-PCR and coat protein (CP) gene nucleotide sequences analysis. In squash plants, antiviral, CMV optical density, biochemical responses, and resistance genes expression of Streptomyces extracts (SE1 and SE2) were performed against CMV by disease incidence, severity (%) assays, ELISA technique, physiological analysis, and real-time quantitative PCR (qPCR). Chemical profiling of the two Streptomyces extracts was investigated using GC-MS analysis. Antiviral activity was performed by curative (C), protective (P), and inactivation (I) techniques under greenhouse conditions. The obtained results of the morphological, biochemical, physiological and molecular level studies of the Streptomycetes isolates ph6 and MARH showed similarity towards the species of Streptomyces and identified as Streptomyces variabilis strain ph6 (OQ283766) and Streptomyces sp. strain MARH (OQ283775). Protective treatments (P: SE1 and P: SE2) resulted in disease suppression of 100%, while curative treatments (C: SE1 and C: SE2) by 87% and 100%, and inactivation treatments (I: SE1 and I: SE2) by approximately 70%, and 80% respectively compared to mock-inoculated plants. The absorbance values of ELISA at 405 nm for P: SE1 and P: SE2 were 0.191 ± 0.02, and 0.187 ± 0.00 respectively compared to CMV-infected plants which recorded 0.854 ± 0.00. Levels of ascorbate peroxidase (APX), catalase (CAT) enzymes, proline content, total photosynthetic pigments, and total phenolic compounds were significantly increased in P: SE1 and P: SE2 treated squash plants compared to CMV-infected plants. Moreover, P: SE1 and P: SE2 increased membrane stabilization and reduced electrolyte leakage. The pathogenesis-related (PR) gene such as PR-b1 was overexpressed by about 3.09-, and 10.37-fold increases and PR-2 by 1.07-, and 1.66-fold increases for P: SE1 and P: SE2 respectively compared to the CMV-infected control group. Chemical profiling of the ethyl acetate extracts of Streptomyces variabilis and Streptomyces sp. MARH authenticated the presence of constituents such as 1,3¬Dinitro¬2-imidazolidinone (42.60%), Nephthoside-1,2',3',4'-Tetraacetate (76.18%), Tetraphenylporphyrinato dichlorotitanium(IV) (12.42%) and L-Lysine (17.01%) respectively. Our results showed that Streptomyces variabilis and Streptomyces sp. MARH are promising strains for production of antiviral natural products, increasing the phenolic compounds, activity of the antioxidant enzymes, and induction the expression of pathogenesis-related genes to generate systemic acquired resistance (SAR) in squash plants. They also supported the potential use of their extracts as an environmentally friendly novel bio-virocides to sustainably stop the spread of plant viruses.