Eradication of needle endophytes
Eradication of needle endophytes was attempted by heating Norway spruce saplings repeatedly in growth chambers. In a preliminary experiment, two 5-year-old healthy-looking saplings were dug up with their roots from a spruce forest nearby Helsinki (Paippinen, Sipoo) in September 2005 and planted into 5-L plastic pots with the initial forest soil attached to the roots and filled up with highly humified peat resulting in a ratio of forest soil to peat to approximately 1:1. The saplings were placed outdoors until onset of the experiment 2 months later. The experiment started by enclosing each sapling pot (including stem base and roots) into an EPS-box (expanded polystyrene) in order to protect roots from high temperature during heat treatments of the shoots. The lid of the box had a small hole corresponding to the sapling stem diameter. The saplings were incubated in a Termaks, KB8400L, growth chamber daily for 8 h at 40 °C in light (15–25 μmol m−2 s−1) and for 16 h at 8 °C in darkness. In this first heat treatment experiment, saplings were heated 8 h per day for 14 days and in the later inoculation experiments 8 h per day for 7 days. Soil temperature within the EPS boxes did not exceed 20 °C. Relative air humidity varied from 10–20% at 40 °C to 25–35% at 8 °C. The heat treatment procedure described above was applied later to 18 other saplings and its suitability was tested, as described under the “Inoculation with needle litter spread below spruce saplings” subheading.
Before and after heat treatment, endophyte presence was investigated from detached needles. Needles of different age classes were treated separately. Current year needles were considered to represent age-class 1 and needles attached to twig segments below the first set of terminal bud scars were considered as age class 2 needles and the next set as age class 3 and so on. Needles were sampled for isolation of endophytic fungi seven times during the experimental time of 14 days. In each time, five needles of both age classes 3 (flushed in 2003) and 4 (flushed in 2002) were removed from each sapling (in total 20 needles per date). Only these age classes were investigated because they were the two oldest age classes providing high enough numbers of needles needed for repeated sampling and because they were presumed to inhabit endophytes at higher frequency than needles of the two younger age classes. Needles were sampled and analyzed for endophyte presence also in some other experiments of this study, and the sampling protocols applied are explained in the descriptions of those experiments.
Determination of endophytic infection frequency
Endophytic fungi, including Lophodermium piceae, were isolated from Norway spruce needles after surface sterilization with sodium hypochlorite as described by Müller and Hallaksela, (1998). The needles were then cut into ca. 2-mm sections (i.e., a piece of needle cut orthogonally to the needle longitudinal direction) which were pushed into water agar in petri dishes (diam. 9 cm). After 4 weeks, incubation at 20 °C endophytic presence was noted as fungal growth from individual needle sections on the water agar plates.
Comparison of three methods to inoculate spruce saplings
Inoculation of Norway spruce saplings was first tried with ascospores obtained from shed needles collected under mature trees in Paippinen, Sipoo in southern Finland (sampling area P, described by Müller and Hallaksela, 1998). The needles were collected using a nylon net (1 × 2 m, mesh size 1 × 2 mm) (see illustration in the Online Resource 1, Fig. 1a). The net was laid on the ground in May 2005, and 4 months later, all needles fallen on the net were removed and checked for mature ascocarps of L. piceae.
Because only few mature ascocarps were found on the needles, we incubated the collected needles under various conditions in order to find out how the development of ascocarps of L. piceae could be triggered. The needles were separated into four groups: (1) green, (2) brown without zone lines or spots, (3) brown with zone lines but without black spots and (4) brown with black spots and with or without zone lines. Additionally, green symptomless needles of age class 5 were removed from eight trees in the Norway spruce forest in Paippinen. The detached needles formed the fifth needle group. After separation into five groups, the needles were stored at 0 °C for 4 months and then used in an experiment in which the needles were incubated in three different conditions. The incubation was carried out in 45 transparent polypropylene containers (12 × 12 × 10 cm, width × length × height) (Sterivent, Duchefa Biochemie, Netherlands) filled first with 200 ml of sand (grain size < 2 mm) on the bottom and thereafter with 1 dl of spruce forest humus. The humus and sand were moistened with deionized water to field capacity; whereafter, 10 ml of water was added. Each of the five needle types were divided into six to fourteen containers (number varied according to availability of each needle class among needles obtained on the nylon net) including 96 to 500 needles per container. Then, the containers were closed with nearly airtight lids and incubated in growth chambers (Termaks, KB8400L) in three different ways. Two to five containers per needle type were allocated to each of the following treatment:
Under visible and UV-light at 5 °C for 4 days and at 0.2 °C in darkness for 3 days each week. Light intensity was 15–20 μmol m−2 s−1 of which approximately 1/3 was in the ultraviolet range.
No light but otherwise similar conditions as in treatment 1.
Temperature continuously at 15 °C and light as in treatment 1 for 9 h per day.
Moisture was added weekly to retain the initial weight of each jar. Needles with ascocarps of L. piceae were counted and removed six times during 250 days. Needles with high numbers of ascocarps were stored at + 0 °C until ascospore extraction 4 days later. Ascospores were extracted by immersing 60 needles, each having at least 2 mature (i.e. opened when wetted) ascocarps, into 100 ml of 0.2% NaCl-H20 and by sonicating this for 1 min. This resulted in a suspension with 83 × 103 ascospores per ml (determined with a Bürker counting chamber).
Twenty millilitres of this suspension was sprayed within the same day when prepared (in June 2006) on two endophyte-free 6-year-old spruce saplings. These saplings had been dug up with their roots from a mature spruce stand nearby Helsinki (Paippinen, Sipoo) in the previous year, transplanted into 5-L containers, heat-treated and placed outdoors. Heat treatment was carried out by heating the saplings during 7 days for 8 h per day in order to eradicate needle endophytes as described previously in the “Heat treatment” section. Each sapling was covered with a polyethylene (0.25 mm) bag to prevent access by spores occurring in ambient air (illustrated in the Online Resource 1, Fig. 1b). Prior to inoculation, 8 needles were randomly removed and weighted before and after wetting which revealed an average retention of 2.2 μg water per needle. This means that well-wetted needles were able to retain up to 183 ascospores per needle. Just before and 2 months after inoculation, the endophyte infection frequency was determined from the three latest needle age classes. For this purpose, six needles per age class (1, 2 and 3), sapling and sampling date (6 needles × 3 age classes × 2 saplings × 2 sampling dates, i.e. in total 72 needles) were randomly removed. Their endophytic infection frequency was determined as described previously in the “Determination of endophytic infection frequency” section.
Inoculation with needle litter spread below spruce saplings
In this field experiment, eighteen Norway spruce saplings representing three clones T4658, T4748 and T5134 were cultivated outdoors covered with polyethylene bags for 4 years as described in detail below.
The saplings were derived from rooted cuttings that were generated by cutting ca. 8-cm-long twig tips from 5-year-old Norway spruce plantlets in February 2006 and cultivated in a mixture of peat and vermiculate at 90–100% air moisture for the first 2.5 months and thereafter outdoors. At the beginning of the experiment in 2008, the saplings were ca. 20 cm high and their needles contained endophytes at a low frequency of 2–18%, depending on the clone. Therefore, the saplings were first heat-treated (during 7 days for 8 h per day) in order to eradicate the endophytic fungi using the method described in the “Heat treatment” section. After the heat treatment, needles were sampled (3 needles per age class including age classes 1, 2 and 3 and per sapling, i.e. in total 3 × 3 × 18 = 162 needles) and examined for endophytic presence (as described in the “Determination of endophytic infection frequency” section) to ensure that endophytes were eradicated. Then, the saplings were transplanted in six groups (each consisting of three saplings, one for each clone) into a shady garden site in Helsinki in October 2008. Each group of three saplings was confined within a 50 × 40 × 80 cm (width × length × height) wooden structure and covered with a polyethylene bag to prevent access by spores possibly driven by the ambient air (as illustrated in the Online Resource 1, Fig. 1b).
Three sapling groups, each including three different spruce clones (i.e. in total nine saplings) were subjected to inoculation with needle endophytes using shed needles collected from the ground of a mature Norway spruce stand. Another similar set of three sapling groups served as controls for the inoculation. The needles used for inoculation were collected with two nylon nets (1 m × 2 m, mesh size 0.2 mm) placed on the forest ground of a mature spruce stand in Paippinen, Sipoo (nearby Helsinki) in November 2008. The needles were removed from the nets in the beginning of May 2009, cleaned from other litter and placed on the ground below the saplings (1 l of needles per each group of three saplings). The needles were wetted with tap water every fortnight from May to July.
In November 2009, needles of each of the age classes 1, 2 and 3 were sampled from the inoculated spruce saplings (three needles per age class and sapling, i.e. 3 needles × 3 age classes × 3 saplings per group × 3 sapling groups, i.e. in total 81 needles) for determination of inoculation success as described previously in the “Determination of endophytic infection frequency” section.
Inoculation with needle litter placed above spruce saplings
This experiment was started after the previous trial (in which needles were spread below the saplings) using the same saplings. All needles below the saplings were removed and “new” needle litter collected under mature spruces from November 2009 to May 2010 and suspended on a metal sieve (mesh size 5 mm) immediately above the saplings at the end of May (as illustrated in the Online Resource 1, Fig. 1c). The needles were wetted with tap water 2 times a week until July.
Infection frequency was determined in November 2010 from 81 needles sampled similarly as described for the previous sampling in 2009. Inoculation showed to be successful because 34–56% of needles of age classes 1, 2 and 3 were infected. In order to achieve a higher infection frequency, this inoculation procedure was repeated by replacing the previous year brown needles collected for inoculation with newly collected ones in May 2011 and May 2012. In November 2012, needles of age classes 1 to 4 were again sampled from the saplings (3 needles × 4 age classes × 3 saplings per group × 3 sapling groups × 2 treatments, i.e. in total 216 needles) and investigated for their endophytic infection frequency as described in the “Determination of endophytic infection frequency” section.
Identification of L. piceae
In the third inoculation experiment (needle litter suspended above spruce saplings), all fungal colonies noted during determination of the endophytic infection frequency in 2012 were isolated and a batch of 30 randomly selected isolates was subjected to identification based on ITS-sequences.
Single hyphal tips from colonies that emerged into water agar from the needle sections (when assessing endophytic infection frequency) were removed with a modified Pasteur pipette and transferred to fresh orange serum agar plates (MOS-agar; Müller et al., 1994). After a 3–4-week cultivation, small agar blocks were cut from each colony edge and transferred into 2-ml cryogenic vials (Nalgene, Merck, Germany) and stored at 6 °C. Later, thirty fungal isolates were randomly selected and cultivated on MOS-agar covered with a cellophane membrane (diameter 8 cm, British Cellophane Ltd., UK) for 2 weeks at 20 °C after which mycelial samples were removed from colony edges and stored at – 20 °C for DNA extraction. DNA extraction, PCR and sequencing have been described previously (Müller et al., 2007). Isolates with ≥ 98% sequence similarity with Finnish L. piceae isolates (described by Müller et al., 2007) were identified as L. piceae.
The colony morphology of all isolates, identified as L. piceae based on their ITS-sequences, corresponded to that of isolates identified by their morphological, chemotaxonomical and genetic characteristics in our previous studies (Müller and Hallaksela, 1998; Müller et al., 2007). The following characteristics of colony morphology, typical for L. piceae on MOS-agar, were used for backing up the molecular identification: slow growth that always ceases before the edge of the dish is reached, colony appearance compact and chewy with a wrinkled or lumpy surface, colony edge uneven, no spore formation and colour highly variable between isolates.
Persistence of needle endophytes in Norway spruce
Persistence of needle endophytes, their occurrence in adjacent needle sections, and the ability of endophytes to grow further within a needle when ageing was investigated by following the endophytic infection frequency for 6 years in needles of saplings protected with plastic bags against further infections by airborne spores as described in detail below.
Five 5-year-old saplings were dug up with their roots from a mature spruce stand near Helsinki (Paippinen, Sipoo) and transplanted in July 2007 into a shady garden site and confined within polyethylene bags (height 1 m, diam. 39 cm, as illustrated in the Online Resource 1, Fig. 1b) in November 2007. Temperature was followed within the bags with a minimum–maximum thermometer. The air temperature never exceeded 28 °C within the polyethylene bags.
Ten needles of those flushed in 2005, as well as ten needles of each of the later age classes, were sampled from each sapling annually in November (from 2007 to 2012) and investigated for endophytic infection frequency after surface sterilization as explained previously in the “Determination of endophytic infection frequency” section. Each colony emerging in water agar from the 2-mm needle sections was recorded according to sapling, needle and needle section to enable keeping track of their initial position along a single needle so that it was later possible to recognize infections occurring in adjacent needle sections. The needle length varied from 6 to 20 mm. All fungal colonies noted during determination of the endophytic infection frequency of needles that were obtained in the last sampling (in 2012) were isolated. From these isolates, 50 were randomly selected for DNA-extraction and sequencing of the ITS-region in order to determine the occurrence of L. piceae (as described in the “Identification of L. piceae” section).
The polyethylene bags did not completely prevent new endophytic infections to emerge in needles since later, some infections were found in needles of 2008 that developed after confining the saplings into bags (data are given in the “Results” section). We assume that older needles became infected by contaminants at a similar intensity as needles of 2008 and subtracted from their infection frequency that observed in needles of 2008.
Results from the eradication experiment of endophytes from spruce needles and from inoculation experiments of spruce needles with L. piceae are reported with figures and in text without statistical analyses. Results from experiments on the persistence of endophytic infections, their occurrence in adjacent needle sections and the ability of endophytes to grow further within a needle when ageing were investigated using statistics described below.
Changes in infection frequencies of L. piceae in needles during the persistence experiment were investigated as follows. First, the infection percentages were corrected for unexpected contamination during the experiment by subtracting the infection percentage found annually in needles of age class 2008 from the respective results obtained for needles of previous age classes 2005, 2006 and 2007. Then, endophytic infection percentages determined annually (until 2012) were compared to results obtained in the first year (2007 or 2008, depending on needle age class) within each age class separately (n = 22 for the needle age class 2005, n = 29 for the age class 2006 and n = 24 for the age class 2007) using linear mixed models (LMMs). Function lme in library nlme in the statistical programme R was used (R Core Team 2017; Pinheiro et al., 2017). The percentage values were logit-transformed for model estimations (Warton and Hui, 2011). Thus, the transformed proportion of needle sections infected was used as a response variable, and as an explanatory variable, we had the sample year (a factor with six levels: 2007, 2008, 2009, 2010, 2011 and 2012, excluding 2007 for needle age class 2007). As a random factor, we had a spruce sapling. Random factor takes into account the fact that needles collected from the same sapling are more similar than observations from randomly collected saplings and needles.
Furthermore, we estimated a generalized linear mixed model (GLMM) to investigate whether the occurrence of adjacent infections (described in the “Persistence of needle endophytes in Norway spruce” section) within a needle increases with needle age, i.e. do individual infections grow with time and occupy a larger portion of aged needles compared to infections in young ones. Only needles with two sections infected were included in the analyses (in total 94 needles) in order to avoid a bias potentially caused by unwanted contaminations during the experiment. The model was estimated using function glmer in library lme4 in R (binomial distribution with logit link function, R Core Team 2017; Bates et al., 2015). As a response we had the occurrence of an endophyte in adjacent needle sections (a factor with two levels: 0 = no endophyte exists in adjacent needle sections, 1 = endophytes existed in adjacent needle sections), and as explanatory variables, we had (1) needle age in years (x – average(x)), (2) squared needle age ((x – average(x))2), (3) cubic needle age ((x – average(x))3) as we assumed a curvilinear response and (4) the number of needle sections investigated per needle. Explanatory variable 4 was included in the model to take into account the fact that the number of needle sections varied from 3 to 6. As nested random factors, we had the code for saplings and the collection year since we assumed that needles within one spruce sapling and the same collection year are more similar than randomly collected needles. All figures were drawn with Excel software (Microsoft, USA).