Four endophytic fungi were isolated from the medicinal plant, Catharanthus roseus, and were identified as Diaporthe spp. with partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS), calmodulin (CAL) genes, and rDNA internal transcribed spacer (ITS) region (TEF1-TUB-HIS--CAL-ITS) multigene phylogeny suggested for species delimitation in the Diaporthe genus. Each fungus produces a unique mixture of volatile organic compounds (VOCs) with an abundant mixture of terpenoids analyzed by headspace solid-phase microextraction (SPME) fiber-GC/MS. These tentatively-detected terpenes included α-muurolene, β-phellandrene, γ-terpinene, and α-thujene, as well as other minor terpenoids, including caryophyllene, patchoulene, cedrene, 2-carene, and thujone. The volatile metabolites of each isolate showed antifungal properties against a wide range of plant pathogenic test fungi and oomycetes, including Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, and Phytophthora cinnamomi. The growth inhibition of the pathogens varied between 10% and 60% within 72 h of exposure. To our knowledge, the endophytic Diaporthe-like strains are first reported from Catharanthus roseus. VOCs produced by each strain of the endophytic Diaporthe fungi were unique components with dominant monoterpenes comparing to known Diaporthe fungal VOCs. A discussion is presented on the inhibitive bioactivities of secondary metabolites among endophytic Diaporthe fungi and this medicinal plant.

Volatile organic compounds (VOCs) from endophytic fungi are becoming a potential antibiotic resource. The inhibitive effects of VOCs produced by an endophytic fungus in Leucaena leucocephala were investigated on plant pathogens in this study. Using standard morphological methods and multigene phylogeny, the fungus was identified as Diaporthe apiculatum strain FPYF 3052. Utilizing a two- compartment Petri plate bioassay method, the VOCs from this fungus showed bioactivity ranging from 23.8% to 66.7% inhibition on eight plant pathogens within 24 hours. The SPME-GC/MS technique identified fifteen volatile compounds with dominant terpenoids γ-terpinene (39.8%), α-terpinene (17.2%), and (-)-4-terpineol (8.4%) from the VOCs. Commercial α-terpinene, γ-terpinene, and (-)-4-terpineol demonstrated inhibition on the tested pathogens at concentrations from 0.2 to 1.0 µl/ml within 72 h in the bioassay system. The inhibition rates were from 28% to 100% percent using 1.0 µl/ml within 48 h. (-)-4-Terpineol was the most active of the terpenoids causing up to 100% inhibition. The data illustrate that these monoterpenes play an important role in the inhibitive bioactivity of the VOCs of D. apiculatum FPYF 3052. Most importantly, (-)-4-terpineol is now for the first time, reported to have capability of strong antifungal activity and could be developed as an antibiotic substance.