ABSTRACT
(1) Background: Insect pathogenic fungi of the genus Metarhizium are under study and in application as highly solicited, more eco-system friendly substitutes for chemical insecticides in many countries and in different agricultural contexts. In Cuba and Florida, Metarhizium strains have previously been isolated from economically important coffee and sugar cane pests. (2) Methods: Unambiguous species delineation within the Metarhizium anisopliae species complex is methodologically challenging. Recently, a species-discriminating PCR approach has been developed based on ribosomal intergenic spacer (rIGS) sequences that covered the prominent four "PARB" species within the complex. This approach is combined here with further genetic markers and is extended to a further species. (3) Results: Metarhizium isolates from Cuba, found to be more naturally associated with the coffee berry borer, Hypothenemus hampei, were morphologically, microscopically and molecular taxonomically characterized. Multilocus sequence analysis based on 5TEF, MzIGS3 and rIGS markers delineated these weevil-associated strains from all previously established Metarhizium species. (4) Conclusions: The isolates under study represent a new fungal taxon proposed to be designated Metarhizium caribense. The rIGS-based species-discriminating diagnostic PCR is a suitable tool for the identification of new Metarhizium species and can be productively combined to approaches using further genetic markers.
ABSTRACT
Trichoderma spp. are filamentous fungi generally observed in nature, which are widely marketed as biocontrol agents. The secondary metabolites produced have obtained special attention since they possess attractive chemical structures with a broad spectrum of biological activities. In Cuba, the species of Trichoderma have been commercially applied for the control of several phytopathogens to protect agricultural crops, but few studies have been carried out to detect and characterize the production of metabolites with biological activity. The strain Trichoderma harzianum LBAT-53 was subjected to an antifungal in vitro assay against Fusarium oxysporum f.sp. cubense by dual culture and volatile metabolite assays and fermented in PDB under constant agitation conditions. The ethyl acetate crude extract was obtained by liquid-liquid extraction. The fungal extract was investigated for the composition of secondary metabolites through chemical screening and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in negative ionization mode. As a result, LBAT-53 showed antagonistic activity in vitro (Class 2) against the pathogen evaluated in direct confrontation (76.9% of inhibition in 10 days) and by volatile metabolites (<40% in 7 days). Furthermore, seven low-molecular-weight phenolic compounds, including chrysophanol, phomarin, endocrocin, and trichophenol A, among others, were identified using UHPLC-ESI-MS/MS. This study is the first work on the characterization of secondary metabolites produced by the commercially applied strain LBAT-53, which is a promising source of bioactive compounds. These results provide a better understanding of the metabolism of this fungus, which is widely used in Cuba as biopesticides in agriculture pest control.
ABSTRACT
(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker.