For. Path. doi: 10.1111/efp.12280 © 2016 Blackwell Verlag GmbH Visualizing the early infection of Agathis australis by Phytophthora agathidicida, using microscopy and fluorescent in situ hybridization By S. E. Bellgard1,4, M. Padamsee1, C. M. Probst1, T. Lebel2 and S. E. Williams3 1Landcare Research Private Bag 92170, Auckland 1142, New Zealand; 2Melbourne Botanic Gardens, South Yarra, VIC, Australia; 3University of Wyoming, Laramie, WY, USA; 4E-mail: [email protected] (for correspondence) Summary Phytophthora agathidicida (PTA) causes a root rot and collar rot of New Zealand kauri (Agathis australis). This study developed techniques to visualize early infection of kauri by PTA in deliberately inoculated seedlings. Conventional light microscopy was carried out on cleared and stained roots using trypan blue to observe PTA structures. Additionally, scanning electron microscopy (SEM) was used to study the PTA root structures at a higher resolution. A fluorescent in situ hybridization assay (FISH) was developed using a PTA-specific probe to label PTA structures in planta. Infection progression in roots of 2-year-old kauri inoculated with PTA at 5, 10, 16 and 20 days post-inocula- tion (d.p.i.) was compared using these three approaches. Light microscopy identified no Phytophthora-like structures in the control treat- ments. In PTA-inoculated plants, lignitubers were produced 5 d.p.i. in cortical cells. Infection was localized after 5 days, but as the infection progressed (up to 20 d.p.i.), the ‘degree’ of root infection increased, as did the number of replicates in which structures were observed. SEM provided higher resolution images; again, no PTA structures were observed in the negative control material examined. The slide-based FISH-specificity assay successfully hybridized with PTA hyphae. Fluorescence was observed using 330–380 nm excitation and an emission filter at 420 nm (DAPI), with PTA nuclei fluorescing a bright greenish-yellow. Cross-reactivity was not observed when the assay was applied to six other non-target Phytophthora species. Successful hybridization reactions occurred between the primer and PTA structures in planta. Applying this FISH assay has allowed clear differentiation of the intracellular and intercellular structures of PTA. The technique can be applied to longer term studies or analysis of ex situ inoculation studies aiming to elucidate differential host-responses to the pathogen. Additionally, the technique could be applied to study the interactions with other fungal endophytes (e.g. mycorrhizal fungi), which could be assessed for biocontrol potential as part of the integrated management of the disease. 1 Introduction Phytophthora agathidicida, the organism formerly known as Phytophthora ‘taxon Agathis’‘PTA’, is the causal agent of a root rot and collar rot of kauri (Agathis australis (D. Don) Loudon) in the northern forests of New Zealand (Weir et al. 2015). The distribution of the disease has now been confirmed throughout the geographic range of the remnant forest (Waipara et al. 2013). The pathogen was first mis-identified as P. heveae, impacting upon kauri in 1971 on Great Barrier Island (Gadgil 1974). Beever et al. (2009) were the first to make the connection between the disorder observed on Great Barrier Island in the 1970s and the ‘dieback’ observed on the mainland in 2006 and that a novel Phytophthora pathogen was involved in the disease. The disorder has been demonstrated to be transferred in soil (Beever et al. 2010) and also via root pieces col- onized by the pathogen (Bellgard et al. 2013). Early infection is facilitated although the fine roots, with the disease pro- gressing along larger woody roots, parasitizing the cambium but rarely invading the xylem. When it reaches the collar, it causes a canker, which is characterized by the production of a resinous exudate associated with parasitism of the cork cambium (Beever et al. 2010). The lesion can extend around the trunk, effectively girdling the tree. A crown decline fol- lows, as a result of the vascular dysfunction, but this takes years to develop, with symptoms including chlorosis, and crown decline (Bellgard et al. 2013). Jung et al. (2013) consider that soilborne Phytophthora species have developed a specific life history that is character- ized by relatively short, active phases of spread via zoospores during periods of wet soil conditions and subsequent pri- mary invasion of healthy tissue. The time course between the primary infection and onset of disease symptoms is not known in the PTA-kauri pathosystem. It is hypothesized that primary infection of healthy kauri roots occurs during peri- ods of favourable environmental conditions, but the nature of the early infection process is unknown as well as the mor- phology of the organism as it infects through the root cortex. Widmer et al. (1998) utilized light and transmission electron microscopy to study the early infection of the fibrous roots of disease tolerant and susceptible citrus hosts by P. nicotianae and P. palmivora. They were able to describe differences in hyphal colonization in the cortex of resistant cul- tivars. This study builds on this earlier work, through the development of a species-specific, fluorescent assay that will preferentially bind to our target pathogen. In addition, the survival strategies and long-term resting structures produced by PTA over time after primary infection are not well understood. This study aimed to compare and contrast three micro- scopic visualization techniques using the homothallic Phytophthora agathidicida and New Zealand kauri as the test system. The specific aims were to: 1. provide a visual description of the infection of 2-year-old kauri plants over a time course of 20 days; 2. develop and test the specificity of a PTA fluorescent in situ hybridization (FISH) assay; 3. describe the infection process using microscopy and the FISH assay. Received: 30.3.2015; accepted: 24.2.2016; editor: T. Jung http://wileyonlinelibrary.com/ 2 S. E. Bellgard, M. Padamsee, C. Probst, T. Lebel and S. E. Williams 2 Materials and methods 2.1 Inoculum production and procedure Inoculation experiments were conducted in a quarantine (Physical Containment [PC], Level 1), naturally lit glasshouse at Landcare Research, St Johns, Auckland. All research at the PC-1 laboratories were carried out under a CTO permit (Biosecurity Act 1993) approving propagation and communication of PTA. PTA inoculum was prepared by growing isolate ICMP 18403 (provided by the International Collection of Microorganisms from Plants, [ICMP]) for 4–6 weeks at 20°C on sterilized millet seeds thoroughly moistened with V8-juice broth (Vettraino et al. 2001; Jeffers 2006). The inoculum was repeatedly rinsed (three times) with sterile reverse osmosis (RO) water to remove unassimilated nutrients before being added to sterile potting mix at a rate of 25 ml per litre of the potting mix. Kauri seed was provided by Dr B. Burns (University of Auckland) from a residence in the West Auckland suburb of Titirangi. The seed was sourced from an open-pollinated tree. Seeds were germi- nated and grown in sterile potting mix for 2 years in the naturally lit PC-1 glasshouse. The temperature in the glasshouse over the 3-week period of the experiment ranged between 18° and 24°C. Mean relative humidity was 70–80%. The same 2-year-old kauri seedlings were transplanted into the inoculated potting mix and then flooded to induce sporu- lation of PTA (Vettraino et al. 2001). There were 20 replicate black, polythene, planter bags (size 1, 600 ml) treated with PTA. Another set of four bags were established as a control using sterilized millet seeds thoroughly moistened with V8- juice broth and incorporated into sterile potting mix at a rate of 25 ml per litre. Bags were arranged in a randomized block design, with PTA-treated plants segregated from negative controls. Plants were watered to field capacity with tap water every other day. The experiment ran from 1 to 20 December 2012. All liquid run off from pot watering was captured and sterilized in an autoclave prior to disposal. 2.2 Progressive harvesting and root segregation Five, 10, 16 and 20 days post-inoculation (d.p.i.), four randomly selected plants were extracted from the potting mix. The used potting mix was disposed of into a quarantine waste bin, which was collected by a certified biological hazard waste disposal company. The shoot height and mass and root length and mass were recorded, and any signs of disease – root necrosis, shoot chlorosis, shoot desiccation, leaf loss/necrosis – were assessed qualitatively (Vettraino et al. 2001). At each of the sampling dates, and at the end of the 20 days, root and collar pieces were surface disinfested in 70% ethanol (EtOH) for 30 seconds and then rinsed in sterile RO water. The root pieces were blotted dry on clean paper towels and plated to Phytophthora-selective agar (Jeffers 2006). The plates were wrapped in foil and incubated at 18°C. Characteristic oospores of PTA were observed in 7 days, and representative cultures were subcultured to fresh Potato Dextrose Agar (PDA) plates for DNA analysis and ITS-sequence confirmation of PTA. Following on from this, the entire root system was fixed in FAA (ethyl alcohol 50 ml, glacial acetic acid 5 ml, formaldehyde (37–40%) 10 ml, RO water 35 ml; Talbot and White 2013). A similar recovery exercise was carried out on the control kauri plants. The individual kauri roots from each sample were carefully segregated into four root classes: root collar, primary roots, secondary roots and tertiary roots. Root metrics were estimated prior to partitioning of the root systems. For a given kauri plant, replicate subsamples of the root system were distributed to four different groups of cryotubes for light micro- scopy, SEM, FISH analysis and a set kept for future RT-PCR analysis. Each of the tube had been supplied with fresh FAA.