Sampling strategyA hierarchical sampling strategy was employed to collect samples (stem
cuttings) from the different locations. Wild material was collected
from seven tropical natural forests: (i) Budongo forest; (ii) Itwara
Central Forest Reserve; (iii) Kalangala (Lutoboka central forest
reserve); (iv) Kibale forest national park; (v) Mabira forest reserve;
(vi) Malabigambo forest and (vii) Zoka forest. In each location
(forest) except Kalangala, samples were collected from five sub-sites
that were separated by distances of at least 5 km. From each sub-site,
five healthy C. canephora trees were identified from which we
collected stem cuttings. Since C. canephora is an allogamous
species, each sampled plant was considered to be genetically unique
and therefore, each sampled tree was regarded as a distinct genotype
in this study. The assumption that each sampled tree is a unique
genotype was confirmed by genetic analysis in Kiwuka et al., (2021).
Contrary to other locations, the Kalangala site comprised remnants of
natural forest systems and secondary forests regenerated from formerly
cultivated coffee fields, and therefore, the coffee populations in
Kalangala were considered wild or feral depending on where they were
collected from. Samples that were collected from natural forest
fragments were regarded as wild while samples from collected abandoned
cultivation fields were considered as feral.
The cultivated samples were collected from two germplasm field
collections of the Ugandan National Agricultural Research Organization
(NARO): National Coffee Research Institute Kituza and from the
National Agricultural Research Laboratories at Kawanda. The cultivated
genotypes were selected on the basis of their historical and passport
data with the aim of representing the total range of traditional and
commercially cultivated C. canephora diversity, including the
two predominant forms found in Uganda: Erecta, or upright forms, and
Nganda, or spreading forms (Thomas, 1935) and the six elite clones,
namely: KW13, KW14, KW15, KW16, KW18 and KW19 (details can be found in
(Kiwuka et al. , 2021)).
Stem cutting establishment
All the collected stem cuttings were rooted in a screen house at the
National Agricultural Research Laboratories (NARL), Kawanda at 0º 25’ N,
32 º 32’ E, 1195 m a.s.l., starting on 30th May 2015.
The establishment of the material from stem cutting followed a tested
protocol by the National Coffee Research Institute (NaCORI,
unpublished). The collected stem cuttings were cut into 7 cm inter-nodal
wood cuttings with one pair of leaves. A total of 7,419 inter-nodal
cuttings, for all the collected genotypes (230) were planted in
poly-pots and placed in transparent plastic cages for root
establishment. The number of cuttings per genotype ranged from 7-99 the
median being 33. The poly-pots had a diameter of 5 cm and a height of 7
cm and were filled with a mixture of topsoil, sand and manure in a ratio
of 3:2:2 by volume. Before planting, each stem cutting was dipped in
rooting hormone (Seradix ’2ʹ, 0.8% w.w, IBA, Twiga Chemicals
Industries, Nairobi, Kenya) to boost their rooting potential. After
seven months, the young plants that had grown from the cuttings were
hardened off and, transferred into 10 L pots. The potting medium
comprised of black loamy forest soil, lake sand and decomposed cattle
manure in the ratio of 3:1:1, with a volumetric water content of 30 %
(± 0.22) at field capacity and 6 % (± 0.16) at permanent wilting point
respectively (See details of the chemical and physical properties of the
potting medium in Appendix Data File A.1.). Ten grams of an inorganic
compound fertilizer comprising: 25% nitrogen, 5% phosphorous, 5%
potassium and 5% of sulphur of the total weight of the elements in the
fertilizer was added per pot. Pots were optimally irrigated for six
months before starting the experimental treatments.
Experimental design
Out of the 230 collected genotypes, 148 produced sufficient number (≥5)
of properly rooted plantlets to start the experiment with. From October
10th to 15th 2016, 16 months after
re-planting the stem cuttings, 1184 rooted plants were arranged into a
split-plot design; with two watering regimes (ample vs restricted-water)
as the main factor and the different C. canephora genotypes as
the sub-factors. Plants were grown in a ‘rain out’ screen house (40 m by
6.5 m) that was blocked into four sections, based on the variation in
radiation that was visually assessed (148 remaining genotypes x 4 blocks
(with each split into two) x 2 water regimes (ample and restricted).
To establish ample vs restricted-water availability treatments, we
assessed the potting medium’s properties, e.g. water content at
field capacity, permanent wilting point and the daily evapotranspiration
rates within the screen house by weighing over time a selection of 10
pots. Soil water loss was also estimated from monitoring soil moisture
content in pots using a soil moisture sensor (Trime-Pico 64/32, HD2 IMKO
Micromodultechnik, Ettlingen, Germany). The ample-water treatment was
set at 25 v% which was about 80% of soil moisture content at field
capacity, while the drought-stressed regime (restricted-water) was
sustained at 10 v% soil moisture at the permanent wilting point.
Plants in the ample-water treatment received on average 1000 ml of water
per watering interval, which was, on average, once a week. Plants in the
restricted-water treatment were subjected to gradually increasing
severity of drought stress and the basic regime was that on average,
plants received 300 ml per week for the first month, 300 ml per
fortnight for the following month, a onetime 300 ml water gift in the
third month and finally a month without water. To minimize the potential
plant-size drought bias i.e., the fact that larger plants consume more
water and are therefore exposed on average to drier conditions, the
following procedure was used: in the initial experimental phase, a
sub-set of plants (54 plants; selected to represent the architectural
[number of leaves, number of primary branches, number of suckers and
leaf area], variation across the experiment) were monitored to
determine their soil water content (both gravimetrically and with the
soil moisture probe) every week and their corresponding number of
leaves, number of primary branches, the number of suckers and leaf area
were non-destructively estimated. Leaf area of fully expanded leaves was
estimated from leaf length and width using the linear model (area per
leaf = leaf length x leaf width x k (k=correction factor = 0.66)) of
Schmildt et al., (2015). These data yielded a correlation between leaf
area and water loss and the relation was used as a guide to determine
the frequency of watering for every plant based on its leaf area. This
procedure ensured that size-dependent effects on the actual soil
moisture experienced by plants were minimized. At the end of the
experiment, it appeared that the amount of water supplied (W (ml)) could
be linearly related to the leaf area (L (cm2)) to each
plant was described by the formula: W = 1479 + 0.178 L ), p
= 0.000 and R2 = 0.27.
The experimental treatment period lasted four months (from plant age 20
months to 24 months; age zero is when the stem cuttings were planted to
root). Data on temperature and relative humidity in the screen-house
were recorded by sensors with data logging (Tinytag logger Plus 2 Dual
Channel Temperature/Relative Humidity, TGP-4500, Gemini data loggers
Ltd., Chichester, Chichester West Sussex, UK) on an hourly basis. The
average daily temperatures and relative humidity of the screen-house
throughout the experimental treatment period were: 23.1 º C (± 4.3) and
83.1 % (± 18.0) respectively while average daily vapour pressure
deficit estimates were 0.49 (± 0.15).
Data collection
Data were collected at three stages: (i) at the start of the treatment
phase; (ii) during the treatment phase and at (iii) at the end of the
treatment phase (Appendix Table A.2.). At the start of the treatment
phase on 25th May 2017 (plant age 20 months) several
non-destructive measurements were done to provide a baseline for later
size increment measurements: plant height, number of nodes, number of
leaves (fully grown and proportion/fractions from estimated full size of
developing ones), length and width of fully expanded leaves and stem
diameter at 5 cm from the base. After these measurements, the youngest
fully expanded leaf pair was marked, to establish a recognition point
for measuring new growth. The second data collection stage (at the point
when 10 % of the plants subjected to drought treatment started to
exhibit leaf wilting (scored visually), was taken 21-24 June 2017. The
final measurement occasion, at the end of the treatment phase, was
conducted at 12-26 September 2017, with measured traits as listed in
Appendix Table A.2.
Methods to measure plant properties
Plant height was measured using a meter ruler from the base (point of
origin from the cutting) to the last node. To estimate area per leaf and
subsequently the total leaf area, we used the same model as that used
for determining leaf area in relation to the watering regimesi.e. we measured length and width and then used the linear model
(leaf length x leaf width x k (correction factor)) of (Schmildtet al. , 2015) on all fully unfolded leaves and obtained a
correction factor (k of 0.66) that was used on all measured leaves. Leaf
area on the main stem was measured in this way for all plants. But due
to the necessity to reduce the workload, the leaf area of primaries and
suckers was measured using the aforementioned linear model, but only for
all plants in one block. For every genotype the leaf area of primaries
and suckers in blocks two, three and four were estimated from the ratio
of leaf fresh weight to leaf area, generated from the measured plants in
block 1. At the end of the experiment, for each plant, leaves were
separated into leaves from the main stem, primaries and suckers. To
obtain total leaf fresh weight (TLFW) and total leaf dry
weight (TLDW), the fresh weight of all leaves was
estimated by weighing fresh leaves while leaf dry weights were measured
after oven drying (70 °C to a constant weight).
Specific leaf area (SLA) was estimated as the ratio of
leaf area and leaf dry weight accumulated within the experimental
treatment phase. The roots of each plant were harvested and cleaned
under running water and on a wire mesh. Using the water displacement
method, fine roots (excluding the taproot with a diameter larger than 3
mm) were dipped in a measuring cylinder to estimate their root volume.
The root volume and total leaf area (TL) of each plant
were used to estimate the root volume to leaf area ratio
(RL). Four growth-related traits were used to
characterize the genotype responses to drought stress. These were
relative growth rate in leaf area (RGRA, see below for
how this was calculated), the total number of leaves
(TNL) total leaf area (TL[cm2]), total leaf dry weight
(TLDW [g]), specific leaf area (SLA[cm2 g-1]) and root volume to
leaf area ratio (RL [cm3cm-2]). Note that all traits, except root volume,
refer to growth during the experimental period, excluding the biomass at
the start of treatments. RGRA was used to assess in more
detail the cultivation status, location and genotype response to
restricted and ample availability of water. Relative growth rates were
used for two reasons: (i) to reduce confounding effects of initial plant
size and (ii) we dealt with very young plants for which the assumption
of them being in exponential growth phase was reasonable. We focused on
area, dry mass and number of leaves because of practical reasons
(measurable non-destructively; base measurements of biomass were not
available) and because leaf area determines light interception capacity,
photosynthesis and subsequent growth (Poorter and Remkes, 1990;
Weraduwage et al. , 2015) (and in coffee fast vegetative growth
are typically associated with high yields (Cilas et al. , 2006)).
RGRA was calculated as