Screening and identification of BP100 peptide conjugates active against Xylella fastidiosa using a viability-qPCR method

Xylella fastidiosa is one of the most harmful bacterial plant pathogens worldwide, causing a variety ofdiseases, with huge economic impact to agriculture and environment. Although it has been extensively studied,there are no therapeutic solutions to suppress disease development in infected plants. In this context, antimicrobialpeptides represent promising alternatives to traditional compounds due to their activity against a wide range ofplant pathogens, their low cytotoxicity, their mode of action that make resistance more difficult and theiravailability for being expressed in plants.Peptide conjugates derived from the lead peptide BP100 and fragments of cecropin, magainin or melittinwere selected and tested against the plant pathogenic bacteria X. fastidiosa. In order to screen the activity of theseantimicrobials, and due to the fastidious nature of the pathogen, a methodology consisting of a contact testcoupled with the viability-quantitative PCR (v-qPCR) method was developed. The nucleic acid-binding dye PEMAXwas used to selectively quantify viable cells by v-qPCR. In addition, the primer set XF16S-3 amplifying a 279 bpfragment was selected as the most suitable for v-qPCR. The performance of the method was assessed bycomparing v-qPCR viable cells estimation with conventional qPCR and plate counting. When cells were treatedwith peptide conjugates derived from BP100, the observed differences between methods suggested that, inaddition to cell death due to the lytic effect of the peptides, there was an induction of the viable but nonculturable state in cells. Notably, a contact test coupled to v-qPCR allowed fast and accurate screening ofantimicrobial peptides, and led to the identification of new peptide conjugates active against X. fastidiosa.Antimicrobial peptides active against X. fastidiosa have been identified using an optimizedmethodology that quantifies viable cells without a cultivation stage, avoiding underestimation or false negativedetection of the pathogen due to the viable but non-culturable state, and overestimation of the viable populationobserved using qPCR. These findings provide new alternative compounds for being tested in planta for the controlof X. fastidiosa, and a methodology that enables the fast screening of a large amount of antimicrobials against thisplant pathogenic bacterium.