Development and evaluation of a real‐time PCR seed lot screening method for Fusarium circinatum, causal agent of pitch canker disease

Abstract

Summary Fusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F. circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F. circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi‐selective medium and identifying it using morphological traits. This method is time‐consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real‐time PCR procedure to screen seeds for the presence of F. circinatum were developed in this study. The real‐time PCR method resulted in the detection of F. circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 × 10−5 ng of F. circinatum DNA per PCR. The culture‐based method detected Fusarium spp. in four of six of the same seed lots. The real‐time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture‐based method requires a minimum of 1–2 weeks. This new real‐time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.

PCR Multiplexes DiscriminateFusariumSymbionts of InvasiveEuwallaceaAmbrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States

“…The newly designed primer sets amplify their correct targets in concentrations at or below 1 ng/ml for all gDNA templates, and at or below 10 pg/ml for three AFC species, which is comparable with previously reported sensitivity for multiplex assays of phytopathogenic fungi (Dreaden et al 2012;Rigotti et al 2002) and bacteria (Balestra et al 2013). The multiplex assays successfully identified AF-3 and AF-4 from pooled DNA from eight batches of freshly macerated E. interjectus and E. validus beetle heads, respectively.…”

“…The spread of Fusarium tree diseases, including pitch canker, which has been confirmed on dozens of native and cultivated pine species and associated with numerous insect vectors, is of great concern to countries such as Australia and New Zealand, where extensive plantations of highly susceptible Pinus radiata are grown (Dick 1998). In response to these threats, several molecular methods for the rapid and sensitive detection of F. circinatum from bark beetles (Fourrier et al 2015), seed (Dreaden et al 2012), and spore traps (Schweigkofler et al 2004) were developed. These molecular tools have likely mitigated additional spread of pitch canker, especially in infected nursery stock, which historically relied on less accurate and less sensitive culture-based screening methods for detection of pathogens, which were prone to false negatives (Dreaden et al 2012).…”

Culture-independent detection and quantification ofFusarium circinatumin a pine-producing seedling nursery

“…(1) TEF-barcode analysis (Geiser et al 2004); (2) H3 PCR-restriction fragment length polymorphism (RFLP) (Steenkamp et al 1999); and (3) PCR with F. circinatumspecific oligonucleotide probes or primers (Schweigkofler et al 2004;Ioos et al 2009;Dreaden et al 2012). Of these, PCR-based methods using specific probes and primers allow for the simultaneous detection and quantification of the pathogen, especially when combined with quantitative real-time PCR (qPCR).…”

A mini-review on the development and emerging perspectives of seed pathology

“…Owing to the high sensitivity and specificity of molecular based seed testing approaches over other known methods (Table 1), numerous PCR-based assays have been reported for seedborne pathogens in the last over 2 decades (Audy et al, 1996;Dreaden et al, 2012;Frederick et al, 2002;Glynn and Edwards, 2009;Wen and Zhang, 2012;Zhang et al, 1999).…”

“…Hence nucleic acid based molecular techniques are being clamoured for in seed health testing, and are being developed (Dreaden et al, 2012).…”