Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

Abstract

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

The Diaporthe sojae species complex: Phylogenetic re-assessment of pathogens associated with soybean, cucurbits and other field crops

“…Although many of the species included in study are well known as pathogens of important crops, the extensive sampling from various geographic locations and different hosts has revealed that D. longicolla and D. sojae can be endophytic in tropical trees and mangroves (Cheng et al 2008;Sebastianes et al 2012;R€ onsberg et al 2013). The closely related taxa D. endophytica, Diaporthe schini, and Diaporthe terebinthifolii were originally described from Schinus in Brazil as endophytes (Gomes et al 2013).…”

Interactions of arbuscular mycorrhizal and endophytic fungi improve seedling survival and growth in post-mining waste

“…Being one of the most dominant endophytes, Diaporthe ( Phomopsis ) species are commonly isolated as endophytes from different dicot plant hosts (Suryanarayanan et al 2002; Sun et al 2011). Some species of Diaporthe , however, show either pathogenic or mutualistic interactions with plants depending on the host and its health; for example, D. phaseolorum is pathogenic to soybean (Santos et al 2011), but endophytic in mangroves (Sebastianes et al 2011). Diaporthe spp.…”

<I>Diaporthe</I>: a genus of endophytic, saprobic and plant pathogenic fungi

“…Curiously, some species of Diaporthe can be either pathogenic or harmless endophytes depending on the host and its health. For example, D. phaseolorum is pathogenic to soybean (Santos et al 2011), but endophytic in mangroves ( Laguncularia racemosa ) (Sebastiane et al 2011). With the deletion of Art.…”