General procedures
All chemicals and media supplies were obtained from Duchefa (Haarlem, The Netherlands) c/o Melford Laboratories Limited (Ipswich, UK) unless otherwise stated. Sterile procedures were performed in a laminar air flow bench with the instruments being sterilised by placing them into a glass bead steriliser at > 250 oC for two or more minutes prior to use, unless otherwise stated. Triple vented 9 cm Petri dishes (type 101VR20) and other plastic ware was purchased from Thermo-Fisher Scientific UK Limited.
Establishment and maintenance of ash shoot cultures
In the autumn of 2012, a batch of open pollinated ash seeds was harvested and pooled from the UKs national collection of ash trees, which consists of over 350 grafted scions collected from across the UK and Ireland, which are held by the Earth Trust at Little Wittenham in Oxfordshire, UK. The origin and layout of this collection of ash trees is fully described by Clark and Webber (2017).
A second seed collection was made in the autumn of 2013, in which seeds from different mother trees were harvested separately with a record of their origin, both of which were used to establish the ash shoot cultures that were developed for this work, as shown in Table 1. Both lots of seeds were stored at 4 oC at the Forestry Commission’s seed store at Alice Holt, Surrey, UK, before being transferred to the FRs Northern Research Station and kept at 4 oC until required. These were used to establish proliferating in vitro shoot cultures as described below, with the separate batches of seeds harvested in 2013 being labelled as half-sib “families”, according to which mother tree they were harvested from.
Table 1 The family sets of proliferating ash shoot cultures used in rooting trials for this study The winged samaras were removed, and the inner seeds soaked overnight in tap water before being surface sterilised by soaking them for 10 min in 2% w/v dichloroisocyanurate, (Sigma-Aldrich Company Limited, UK) plus 0.02% v/v Tween 20 (Sigma), before rinsing them 3× in sterile water. Intact embryos were dissected from the seeds and placed into 9 cm Petri dishes containing 30 ml of hormone-free DKW medium with standard vitamins (i.e. DKW0) plus 2 mg/L Pyridoxine (Driver and Kuniyuki 1984). The medium also contained 9 g/L Plant agar and 30 g/L sucrose, and was adjusted to pH to 5.8 with KOH before being autoclaved at 121 oC for 15 min.
The embryos were kept in these conditions for 2 weeks until they germinated, and were then transferred into 300 ml glass jars with screw-top lids, containing 50 ml DKW0 medium, under the same conditions for another 4 weeks. The culture conditions were a 16 h day/8 h night cycle at 25 oC ± 1.5 oC with 50 μM.m2.s−1 of photosynthetically active light at shelf level from 125 W Thorn Natural Daylight fluorescent lamps. The embryos were allowed to grow until they were 5–8 cm high, after which they were used for providing the explants for generating the shoot cultures used in the experiments described in this paper.
Hypocotyl segments (1–3 cm in length) were dissected from the seedlings and laid sideways into 9 cm Petri dishes containing 30 ml of DKW3 medium (i.e. DKW0 medium with 3 ppm BAP added), to induce shoot cultures. Larger callus pieces or shoots were transferred to 300 ml glass jars with 50 ml of DKW3 medium, solidified with 9 g/L of Plant Agar.
Shoot apices from these dissections were placed upright into DKW3 medium as it was found that many of them could convert into shoot cultures if left long enough. Other media permutations were trialled, including DKW5 medium with 5 ppm BAP added, as well as woody plant medium (Lloyd and McCown 1980) with the same vitamins and hormones as for the DKW media, but the results were worse than that observed with DKW based media (data not shown), and so this was not pursued further. The callus or shoots that were generated, were subcultured onto fresh DKW3 every 4–6 weeks, when the shoots were 3–5 cm long.
Rooting and weaning of ash plantlets
To induce rooting in the proliferating ash shoots, in-vitro proliferating shoot pieces 3–5 cm in length were excised and placed basally ~ 1-cm deep into a 300-ml glass honey jar, containing 50 ml of DKW medium supplemented with 3 ppm IBA and no BAP. The shoots remained on this medium for 2 weeks under the standard culture conditions and were then moved to DKW0 medium in jars for a further 4–6 weeks also under the same conditions, with 4 shoots per jar and 12 shoots per treatment per clone. After this time the plants were scored as alive or dead and for the formation of any roots in vitro, after which any survivors were transferred to the nursery. The aim was to assess at least 10 shoot clones per family, but as the number of clones produced for each family varied, so between 7 and 12 clones were actually tested at least twice for each family in most cases.
The plantlets were potted by removing them from their culture vessels to a dish of tap water, and then placed into modular trays, containing ~ 150 ml compost per pot (Levington Advance, low nutrient seed and modular compost, ICL, Ipswich, UK), and watered in. Batches of plants were placed into 55 cm × 30 cm weaning trays with transparent lids with adjustable vents in a temperature-controlled glasshouse. The glasshouse was set to 20 oC ± 2 oC day and night, and 16 h of daylength, supplemented from LEDs providing 400 μM.m2.s−1 of photosynthetically active light at shelf level if the ambient light fell below this level, with the humidity set to 60% rh. The vents of the weaning trays were not opened until the majority of the ash plantlets within each tray had started to grow, usually 4−5 weeks after the plantlets had been potted. Two to three months later, the surviving ash plants were re-potted in 1 L pots filled with the same compost and moved outside.
Plant scoring and statistical analysis
The individual shoots were scored for whether or not they had (i) survived the in vitro root induction process, and (ii) produced one or more observable roots in vitro. Several clones at a time were tested in batches as previously described and depending on the availability of shoot material, with the performance of each clone being tested at least twice in different trials. It was also recorded whether shoots were contaminated by visual or molecular assessment, before and after rooting was induced. Unless otherwise stated, all the results reported here are for the uncontaminated clones. The survival of the plantlets was assessed again one month after their transfer to the nursery and occasionally thereafter.
The effect of contamination on the shoots survival p, was assessed using a binomial generalised linear mixed model (GLMM) with a logit link accounting for over-dispersion as follows:
$${\displaystyle \begin{array}{c}p\sim Bin\left(n,p\right)\\ {} logit\left({p}_{ij}\right)=\mu +{\alpha}_i+{\theta}_{ij}\end{array}}$$
Where n is the number of seedlings in each trial; μ the mean proportion surviving; α is the fixed effect of contamination status; θij~N(0, σ2) is a random effect with one level for each observation to account for overdispersion and \(logit(p)=\mathit{\log}\left(\frac{p}{1-p}\right).\)
The proportions surviving the nursery and in vitro stages were modelled in a similar manner using binomial GLMMs with rooting status treated as a fixed effect, with family; clones; and trials treated as random effects. The models were checked for over-dispersion, but no correction was necessary. The Family effect was checked using a likelihood ratio test. Analysis was carried out using the package lme4 version 1.1–21 (Bates et al. 2015) in R 3.6.1 (R Core Team 2019). The plant rooting and survival scores, graphical presentations of all the statistical analyses, and supporting metadata documents are available as supplementary files (Fenning 2022) to this paper via the public data repository site Zenodo which can be accessed via the link https://doi.org/10.5281/zenodo.6037429 and https://zenodo.org/record/6257888. Additional explanations are also available upon request to the corresponding author.
Identification of a bacterial contaminant
A creamy/pink contaminant was observed on many of the ash tissue cultures, which was isolated and subcultured onto DKW0 media. Repeated efforts to eliminate it were made by incorporating 100 μg/ml each of the filter sterilised antibiotics cefotaxime, augmentin, and vancomycin, into the DKW3 media after it had been autoclaved. Plant material on this medium was subcultured weekly to ensure that the antibiotics were maintained at an effective level. To identify this organism, DNA sequencing and PCR methods were employed at SASA as described below, with the ash samples being extracted at FR.
A 10-min boiling method was used for the bacterial samples with a conventional PCR carried out using universal bacterial 16S primers 27F/1492R (Heuer et al. 1997) and a cycle with initial denaturation at 94 oC for 5 min, followed by 35 cycles of denaturation at 94 oC for 1 min, annealing at 55 oC for 1 min and extension at 72 oC for 2 min, with a final extension at 72 oC for 15 min. Agarose gel electrophoresis confirmed a product of expected size. Sanger sequencing of the samples (3500xL Genetic Analyzers with 50 cm arrays POP-7, Applied Biosystems, Life technologies) was performed using the same primers. Sequences were visualised with Geneious 9.1.8 and run through BLAST and identified as B. megaterium or possibly B. aryabhattai.
The 16S regions are similar between related bacterial species, so to differentiate between them, further sequencing using the recombinase A (recA) primers of Mohkam et al. (2016) was performed. DNA was extracted from seven closely related Bacillus species, cultured on nutrient agar: B. megaterium, B. aryabhattai, B. altitudinis, B. flexus (from DSMZ, Germany) (https://www.dsmz.de/), B. subtilis, B. pumilis, and B. amyloliquefaciens (from SASA), using a chloroform-isopropanol method with Proteinase K added (20 mg/mL) (Reid et al. 2009). DNA from the ash samples was extracted at FR by freezing them in liquid nitrogen, then grinding in a Retsch mixer mill for 1 min using two 3 mm steel ball bearings, followed by a Qiagen Dneasy plant kit.
Based on the resulting recA sequences, new sets of primers and probes were designed for regions with high inter-species polymorphism, following criteria from Integrated DNA Technologies (IDT) using Geneious 9.1.8, and tested for their specificity to the contaminant (Table 2 in Appendix). Primers/probes sets were ordered from Eurofins Genomics (Reporter: FAM; Quencher: BHQ). In vitro real-time PCR assays were performed to validate these primers/probes, using a final primer concentration of 0.5 μM and a standard qPCR cycle (on 7900 HT Fast Real-time PCR System, Applied Biosystems, Life technologies), with a 2-min hold at 50 oC, an initialisation for 10 min at 95 oC, followed by 40 cycles of denaturation at 95 oC for 15 s and annealing/extension at 60 oC for 60 s.
Validation tests were performed to assess the quality of the primers, with each set of primers/probes tested against the seven Bacillus species, along with monitoring the effect of diluting the DNA extracts: 0.05 ng, 0.1 ng, 0.5 ng, 1 ng, and 5 ng after quantifying DNA samples on a Nanodrop (NanoDrop-1000 Spectrophotometer, Thermo-Fisher Scientific UK Limited). All DNA samples were tested with the newly designed primers/probes using two qPCR machines: QuantStudio Flex 6 and 7900 HT (Fast Real-time PCR System), both by Applied Biosystems (Life technologies). Finally, the origin of the samples was tested by evaluating amplification of leaf DNA extracts from five trees plus and 6 seeds from ash trees growing in the Bush Estate near FR-NRS, as well as from ash shoot cultures, representing a variety of contamination states. To validate the recA primers as a PCR tool for contamination identification, DNA samples from known contaminated shoot cultures were diluted (1:100) before being re-tested. The PCR primers of Nayak et al. (2013) for amplifying the PhaC gene were tested twice against the seven Bacillus species investigated. Additional data about the PCR conditions used can be found via the public data repository site Zenodo, which can be accessed via the link https://doi.org/10.5281/zenodo.6037429. The presence of the contaminating bacteria in the plant samples tested by these means was simply scored in a ‘plus’ or ‘minus’ manner, without any attempt being made to use the PCR methods so deployed to quantify their presence in the samples. All the shoots of an individual clone were considered as being ‘contaminated’, even if only one shoot within a clonal batch was observed to be affected.