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Real-Time PCR for Detection and Quantification, and Histological Trees DOI 10.1007/s00468-015-1350-9 ORIGINAL ARTICLE Real-time PCR for detection and quantification, and histological characterization of Neonectria ditissima in apple trees 1 2 3 4 Marjan Ghasemkhani • Anna Holefors • Salla Marttila • Kerstin Dalman • 1 3 6 5 Anna Zborowska • Mira Rur • Jonathan Rees-George • Hilde Nybom • 6 7 1 Kerry R. Everett • Reiny W. A. Scheper • Larisa Garkava-Gustavsson Received: 10 June 2015 / Revised: 7 November 2015 / Accepted: 28 December 2015 Ó Springer-Verlag Berlin Heidelberg 2016 Abstract pathogen identification. A quantitative real-time PCR Key message We designed a pair of primers from a (qPCR) assay was developed for both detection and region of the b-tubulin gene to detect and quantify quantification of this pathogen in infected apple cultivars. Neonectria ditissima in wood of some infected apple Several primer sets were designed from regions of the b- cultivars, and optimized light microscopy to study tubulin gene. One primer set passed several validation fungal-plant interactions. tests, and the melting curve confirmed species-specific Abstract Neonectria ditissima, the causal pathogen of amplification of the correct product. In addition, the N. fruit tree canker, is a sordariomycete fungus that affects ditissima biomass could be detected at variable amounts in apple orchards, especially in north-western Europe. To samples from the infection sites of six different cultivars, prevent serious disease epidemics, an accurate, rapid, and with ‘Aroma’ having the lowest amount of N. ditissima sensitive method for detection of N. ditissima is needed for biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (par- Communicated by W. Osswald. tially resistant) was used in a histopathology study. In both Electronic supplementary material The online version of this detached shoots and trees, fungal hyphae were found in article (doi:10.1007/s00468-015-1350-9) contains supplementary cells of all tissues. No qualitative differences in the anat- material, which is available to authorized users. omy of the infected samples were observed between the & Marjan Ghasemkhani cultivars. In the detached shoot experiment, both cultivars [email protected] were affected but differences in the rate of disease pro- gression suggest that the partially resistant cultivar could 1 Department of Plant Breeding, Swedish University of Agricultural Sciences, Box 101, 230 53 Alnarp, Sweden resist the fungus longer. The qPCR assay developed in our 2 study produced reproducible results and can be used for In vitro Plant-tech AB, Geijersgatan 4, 216 18 Limhamn, N. ditissima Sweden detection of in infected trees. 3 Department of Plant Protection Biology, Swedish University Keywords Apple canker Anatomy B-tubulin gene of Agricultural Sciences, Box 102, 230 53 Alnarp, Sweden Á Á Á Neonectria galligena Á Nectriaceae Á qPCR 4 Department of Chemistry and Biotechnology, Uppsala Biocenter, Swedish University of Agricultural Sciences, Box 7026, 750 07 Uppsala, Sweden Introduction 5 Department of Plant Breeding, Swedish University of Agricultural Sciences, Balsga˚rd, Fja¨lkestadsva¨gen 459, 29 194 Kristianstad, Sweden Neonectria ditissima (Tul. & C. Tul.) Samuels & Rossman, 6 The New Zealand Institute for Plant and Food Research Ltd, previously known as Neonectria galligena (Bres.) Rossman PB 92169, Mt Albert, Auckland, New Zealand & Samuels, is a fungal pathogen that occurs in almost all 7 The New Zealand Institute for Plant and Food Research Ltd, apple-producing areas of the world, e.g., Europe, Asia, the PB 1401, Havelock North, New Zealand Middle East, North America, South America, and New 123 Trees Zealand (CPCI 2014). This pathogen causes cankers on hybridization probe, designed to the ITS1 region. A mag- numerous tree and shrub species, and is especially preva- netic capture-hybridization PCR technique (MCH–PCR) lent in commercial apple (Malus 9 domestica) and pear employing a biotinylated capture oligonucleotide could (Pyrus communis) orchards (Castlebury et al. 2006). The detect fungus in old cankered tissue and in asymptomatic pathogen attacks branches and twigs to cause dieback in tissue either adjacent to a canker or in a newly infected full-grown trees. The lesions often expand to girdle the apple tree whereas PCR without MCH was unsuccessful main trunk and kill all branches above the infected point. In (Langrell and Barbara 2001). However, application of addition, this fungus can cause rotting at the calyx end of MCH-PCR can be limited by the additional cost and rela- fruits (eye rot). Symptoms on fruit are generally expressed tive assay complexity. By comparison, qPCR is technically during prolonged storage causing a serious post-harvest straightforward. problem (Xu and Robinson 2010). During the early stage of qPCR assay development in Since N. ditissima can produce spores throughout the the present study, specific ITS1 region primers Ch1/Ch2 year and infect through suitable entry sites (leaf scars, used by Langrell (2002) for detection of N. ditissima in emerging buds, wounds due to tree ties or pruning), control end-point PCR, were tested in qPCR. The region targeted of this disease is difficult (Webster et al. 2001). Trees can by these primers apparently did not contain a sufficient also become infected during propagation and the infection number of well-positioned polymorphisms to differentiate may remain latent for 3–5 years before symptoms appear between N. ditissima and apple since apple DNA was (McCracken et al. 2003). amplified in samples from tissue-cultured plants. More- Canker symptoms caused by N. ditissima vary depend- over, these primers generated a large product (412 bp) ing on the stage of disease development, age of the infected whereas short amplicons are essential to obtain reasonable wood, and cultivar (Gustavsson 2012). On twigs, cankers amplification efficiencies in qPCR (Kubista et al. 2006). caused by N. ditissima can be confused with those caused Therefore a target DNA region, the b-tubulin gene, was by Neofabraea malicorticis H. S. Jacks. (syn. Cryptospo- instead selected and new primers were designed. Crous riopsis curvispora (Peck) Gremmen) (Braun 1997). Tra- et al. (1999) showed that the b-tubulin gene is more ditionally, microscopic examination of spore size and informative than the ITS regions for distinguishing some shape has been used to differentiate between the canker species of ascomycetous fungi, e.g., Cylindrocladium. The causative pathogens (De Jong et al. 2001; Gariepy et al. b-tubulin gene region is highly conserved in eukaryotes 2003; Kasson and Livingston 2009). and has proved useful for designing species-specific pri- The ability to detect and quantify fungal DNA using mers in previous studies (Nakayama et al. 2013; Yun et al. real-time, or quantitative PCR (qPCR), has been developed 2013). The b-tubulin gene contains an extremely variable and applied to plant pathogens (Catal et al. 2013; Haegi 50 end and a more conserved intron-poor 30 end, and has et al. 2013). qPCR thus provides new research opportuni- been used to determine levels of relatedness in ascomy- ties to study the diagnosis and epidemiology of fruit tree cetes (Landvik et al. 2001). A paralogous copy has been canker (Gadkar and Filion 2014). discovered in ascomycetes (Gold et al. 1991; Panaccione An oligonucleotide primer (ChInt), used in combination and Hanau 1990) but the duplication event has apparently with the universal ITS4 primer, has previously been occurred after the divergence of the sordariomycetes designed from the internal transcribed spacer (ITS) 1 (Landvik et al. 2001; Zhao et al. 2014). region of ribosomal DNA (rDNA) of N. ditissima (Brown More information on the plant–pathogen interaction is et al. 1993). However, the application of this primer set is needed for understanding how partial resistance to apple limited since it cannot differentiate between closely related canker can arise in some apple cultivars. In general, host species which co-exist on the same host (Langrell 2002). plants defend themselves by either structural characteristics Langrell (2002) designed an improved pair of primers or biochemical compounds to inhibit pathogens from (Ch1/2) to detect N. ditissima, also from the ITS regions of gaining entrance and spreading within the plant. In a his- the ribosomal RNA gene. This primer set was mostly tological study in Fraxinus mandshurica (Manchurian ash), successful in detecting N. ditissima in cankered wood but, Sakamoto et al. (2004) showed how N. ditissima affected on occasion, the reaction was inhibited by contaminating the stem tissues during canker formation; fungal hyphae substances from lignified wood resulting in false negatives. were found in different cell types, including the vessel By contrast, the fungus could not be detected in symp- lumina of the affected xylem. Previous studies reported tomless tissue immediately adjacent to cankers, probably significant differences between cultivars in the level of because the PCR was not sufficiently sensitive or was resistance to N. ditissima and several cultivars like ‘Cox’s inhibited by contaminating substances. Langrell and Bar- Orange Pippin’, ‘Elise’, ‘James Grieve’ were found to be bara (2001) improved the PCR sensitivity and reliability very susceptible, while ‘Santana’, ‘Aroma’, ‘Florina’ through implementation of a specific complementary showed relatively high level of partial resistance (Garkava- 123 Trees Gustavsson et al. 2013; Ghasemkhani et al. 2015). How- ITS sequencing of Neonectria ditissima isolates ever, comparative studies on the invasive behavior of the fungus on susceptible versus partially resistant hosts have ITS1F and ITS4 (TAG Copenhagen) were used to amplify not yet been conducted. the internal transcribed spacer (ITS) region of the ribosome In this study, a qPCR assay was applied to identify and encoding genes (White et al. 1990; Ihrmark et al. 2012). quantify N. ditissima in cankered apple trees, and investi- PCRs were performed in a 2720 Thermal Cycler (Applied gate a possible association with levels of partial resistance Biosystems) with 12.5 ng of DNA of the isolates from of the hosts.
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