13C labeling at the whole-tree level
We conducted whole-tree-crown 13C labeling experiments
in summer 2017, i.e. in the 15th year of irrigation
treatment (Joseph et al., 2020). Six mature pine trees that were
severely infected (over) by mistletoes (three control & three
irrigated) were selected from the labeling experiment for the present
study. For each selected tree, the whole tree crown was enclosed within
a large temperature controlled transparent chamber, and approx. 10 g of
CO2 with >99 atom% 13C
(Cambridge Isotopes, Tewksbury, MA, USA) was released into the chamber
over a period of 3.5 h. Pulse labeling of the six trees was applied from
29 to 31 August 2017 (one pair of trees (control / irrigated) per day).
Within this whole-tree labeling experiment, we conducted the following
two experiments for the present study (Fig. 1):
Wrapping experiment (Exp. 1) : We selected six to eight
mistletoe clusters from each of the six 13C-labeled
trees. Half of them (three to four) were randomly selected and wrapped
with gas-tight plastic sheets to avoid direct uptake and assimilation
of 13CO2, and additionally darkened
with aluminum foil to avoid exposure to sunlight before the chamber
was closed (Fig. 1). The other three to four clusters selected per
tree remained unwrapped and were allowed to take up13CO2 during the 3.5 h of labeling.
Tissues from the host (needle, twig xylem and phloem) and mistletoe
clusters (leaf and shoot) were sampled at -1 d (the day before
labeling) and at 4 h, 8 h, 24 h, 3 d, 7 d, 14 d, 30 d, 60 d, and 180 d
after the start of labeling for analysis of the 13C
abundance.
Removal experiment (Exp. 2) : In each of the six13C labeled trees considered in this study, three
well-foliaged branches (around 1 m in length) that were infected by
mistletoes were selected for a girdling treatment to stop the
basipetal transport of photo assimilates (Fig. 1). A bark strip
(including the phloem) of 2 cm width was removed over the entire
circumference of the branch, basipetal to the mistletoe, 1 d before
the whole-tree labeling started. In one of the three girdled branches
per tree, all mistletoe tissues (leaf and shoot) were completely
removed before the 13CO2-labeling
(mistletoe removal), while in a second girdled branch, all host
needles were removed (needle removal). The third one was kept intact
and was used as a control (no removal). Plant material from the host
(needle, twig xylem and phloem) and mistletoe (leaf and shoot) were
sampled at -1 d (the day before labeling) and at 4 h, 8 h, 24 h, 3 d,
8 d, and 15 d after the start of labeling for 13C
abundance measurements. Measurement of non-structural carbohydrate
(NSC) concentrations was conducted for the sampling at -1 d, label1 d,
3 d, 8 d, and 15 d.