Plant material and experimental design
In order to test if water stress can be used as a priming factor for improving avocado
tolerance to R. necatrix, a ‘cross-factor priming’ experiment was carried out in 2017 at the Institute of Agricultural Research and
Training (IFAPA) (Málaga, south-eastern Spain, 36º 40' 25'' N, 04º 30' 11'' W, elevation
of 32 m below sea level). One hundred and twelve 2-years old clonal ‘DusaTM’ plants propagated by Brokaw nursery (Brokaw España S.L.) using a modified Frohlich
method , were grown in 16L pots containing a sterilised mixture of organic substrate
and sand supplemented with a slow release fertilizer (Basacote Plus 6M, Compo Expert
‘Dusa’ plants were kept in a greenhouse under day light illumination and semi-controlled
conditions of air temperature (T) and relative humidity (RH). Photosynthetic photon
flux density (PPFD), T and RH conditions inside the greenhouse were continuously registered
by a quantum sensor (Apogee SQ-110, USA) and by a T/RH U23-001 HOBO® Pro v2 logger (Onset Computer Corporation, USA). Maximal midday values of PPFD varied
between 440 and 1012 mol m−2 s−1, and daily T was allowed to fluctuate according to external weather conditions,
but its variation range inside the greenhouse was maintained between 20±10ºC by an
automatic cooling system and heating when necessary. The RH values inside the greenhouse
were always over 40%.
The experimental design is depicted in Fig. 1. At the beginning of the experiment
(t0), plant physiological status was tested non-destructively by measuring chlorophyll
fluorescence at predawn. Plants were distributed in two sets of 56 plants to conduct two trials. On each trial,
18 plants were randomly assigned to a control group, in which soil moisture was maintained
at field capacity (Fc) throughout the experimentation, and two sets of 19 plants were
subjected to controlled substrate drying-up until they reached 50% of Fc (i.e. mild
water stress, mild-WS) and 25% of Fc (i.e. severe water stress, severe-WS), respectively.
Once these soil water content levels were attained (after ~16-17 days; t1), full irrigation was restored in all plants and drought recovery response was assessed
one week after re-watering (i.e. after ~23-24 days; t2). Hereinafter, the term ‘primed plants’ refers to plants subjected to each of the water stress levels followed by a recovery
period. The pathogenicity test with R. necatrix was performed at t2 as described below.
Soil moisture was monitored with a wet sensor (HH2 Moisture meter, Delta-T Devices.
Cambridge, England), previously calibrated for the substrate, which also allowed to
adjust the level of volumetric soil moisture (v/v) on each water treatment (mild-WS
and severe-WS) in relation to the soil water holding at field capacity (Fc~0.4 v/v).
Once a week, plants were fertilised with an NPK solution (Kristalon Blue 17-6-18,
Yara, UK) supplemented with iron chelate (Sequestrene®, Syngenta, Spain).
Along the experimentation, physiological measurements and root samplings were carried
out at t1 and t2. Roots were sampled from plants others than those used for inoculation.
Midday (12:00-14:00 am) leaf water potential was measured at t1 (when mild-WS and severe-WS plants reached 50% and 25% of Fc) and at t2 (one week after re-watering) using a Schölander pressure chamber (model 3005; Soil
Moisture Equipment Corporation, Santa Barbara, CA, USA). Measurements were done in
one mature fully developed leaf per plant close to the main stem. After cutting, leaves
were immediately placed in the chamber following the recommendations made by Hsiao
Relative leaf water content (RWC), the specific leaf mass area (LMA) and relative
chlorophyll content (SPAD index) were measured only at t1. For RWC determinations, leaf discs (2 cm2) were sampled at midday, weighed to obtain fresh weight (FW) and immediately imbibed on distilled water for 24 h at 5ºC in darkness for obtaining
turgid weight (TW). Afterwards, samples were oven dried at 80ºC for 48h to get dry weight (DW). RWC was calculated as follows:
RWC (%) = [(FW – DW) / (TW – DW)] x 100
The specific leaf mass area (LMA) was calculated as the ratio between disc dry weight
and disc area (g cm-2).
The SPAD index was non-destructively measured at midday on one leaf per plant by a
hand-held SPAD 502 meter (Minolta, Osaka, Japan). This index provides an estimation
of leaf chlorophyll content consistent with leaf greenness . On each plant, averaged
SPAD values were calculated from three readings per leaf.
In vivo chlorophyll a fluorescence signals were measured with a portable fluorometer PAM-2100 (Heinz Walz,
Effeltrich, Germany) at predawn (at t0) and midday (at t1 and t2) in one leaf per plant. The so-called saturation pulse method was used to determine
all fluorescence parameters . Dark-adapted parameters (i.e. minimal fluorescence
(F0), maximal fluorescence (Fm) and maximal photochemical efficiency of PSII (Fv/Fm=[Fm−F0]/Fm) were determined at predawn (05:00-07:00 am). The steady-state fluorescence (Ft), maximal fluorescence (Fm’) and minimal fluorescence yield of a pre-illuminated sample (F0’) were assessed in light acclimated leaves (∼450 µmol quanta m−2s−1). The relative quantum yield of PSII photochemistry (ΦPSII=[Fm’−Ft]/Fm’) , the fraction of PSII centres in open state (qL)  and the extent of “Stern-Volmer” non-photo-chemical fluorescence quenching
(NPQ=[Fm−Fm’]/[Fm’])  were calculated.
Leaf gas exchange was measured at midday (11:00-14:00 am) at t1 and t2 in one mature exposed leaf per plant. Measurements were performed with an open portable
photosynthesis system (model LI-6400, LI-COR, USA) equipped with a LED-light source
(6400-02B), coupled to a sensor head/IRGA, and with a CO2 mixer (6400-01) to modify the incoming air’s CO2 concentrations. Operating flow rate was 500 mL min–1 and CO2 partial pressure was 400 ppm. Saturating photosynthetic photon flux density (1000
µmol m–2 s–1) was chosen as default condition. Leaf temperature was kept at ∼20ºC and relative humidity was adjusted to 50% (vapor pressure deficits ∼1.4 kPa). Net CO2 assimilation rates (AN) and stomatal conductance (gs) were estimated with the equations of Von Caemmerer and Farquhar .
Avocado roots from control, mild-WS or severe-WS were harvested at t1 and t2 in plants others than those used in the pathogenicity test. Three biological replicates
were used for RNA extraction. RNA from ground root tissue was extracted using the
CTAB extraction method , a simple and efficient method for isolating RNA from
pine trees with slight modification. The chloroform:isoamyl alcohol step was repeated
3-5 times, depending on the stability of the interphase and colour of the sample.
RNA quantity and quality were determined based on A260/280 and A260/230 wavelength
ratios using a NanoDrop® ND-1000 (Nanodrop Technologies, Inc., Montchanin, USA) spectrophotometer.
RNA integrity was confirmed by the appearance of ribosomal RNA bands and lack of degradation
products after separation on a 2% agarose gel and Red Safe staining. DNase treatment
of RNA was performed by the addition of 1 U RNase-free DNase (Thermo Scientific, Life
Technologies Inc., Carlsbad, California, USA), 1 μL 10x reaction buffer with MgCl2, 1 μg RNA, 0.5 μL of RiboLock RNase Inhibitor (Thermo Scientific Inc., California,
USA) and diethylpyrocarbonate-treated water to a final volume of 10 μL. The mixture
was incubated at 37 °C for 45 min followed by the addition 1 μL of 50 mM EDTA and
incubation at 65 °C for 10 min.
Quantitative Real-Time PCR
Single stranded cDNA was synthesized using iScript Reverse Transcription Supermix
(Bio-Rad Laboratories Inc., California, USA) according to manufacturer´s instructions.
The cDNA was analysed for genomic DNA contamination by PCR using gene specific primers
F3H-F (5´-TCTGATTTCGGAGATGACTCGC-3´) and F3H-R (5´-TGTAGACTTGGGCCACCTCTTT-3´), which
flank an intron of the eflavone 3-hydroxylase (F3H) gene. PCR amplifications were
carried out as previously described by Engelbrecht and van den Berg  using first-strand
cDNA as the template.
The expression of thirteen avocado genes was investigated. The actin gene was used
as endogenous control for normalization. Primers sequences for endogenous control
gene and the thirteen avocado genes are presented in Table S1. Primer pairs were chosen
to generate fragments between 70 to 140 bp and designed using Primer 3 software (
http://bioinfo.ut.ee/primer3-0.4.0/, [114, 115]). Primer specificity was tested by first performing a conventional PCR
and confirmed by the presence of a single melting curve during qRT-PCR. Serial dilutions
(1:10, 1:20, 1:50, 1:200) were made from a pool of cDNA from each treatment and time-points,
and calibration curves were performed for each gene. For qRT-PCR, the reaction mixture
consisted of cDNA first-strand template, primers (500 nmol final concentration) and
SYBR Green Master Mix (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad) in a total
volume of 20 µl. The PCR conditions were as follows: 30 s at 95ºC, followed by 40
cycles of 15 s at 95ºC and 30 s at 60ºC, 3 min at 72ºC, 1 min at 95ºC. The reactions
were performed using an iQ5 real-time PCR detection system (Bio-Rad). Relative quantification
of the expression levels for the target was analysed using the comparative Ct methods
. All reactions were done in triplicate.
Pathogenicity test in avocado plants
Inoculum was produced on wheat seeds according to Sztejnberg and Madar . Briefly,
seeds were soaked for 12 h in 250 ml Erlenmeyer flasks filled with distilled water.
The flasks, each containing 100 g of seeds, were subsequently autoclaved after excess
water drained off. After sterilization, four fungal discs of a 2-week-old culture
of R. necatrix grown on potato dextrose agar (PDA) were placed aseptically in each flask and incubated
at 24°C in the dark for three weeks until wheat grains were homogeneously covered
by R. necatrix mycelium. Seven days after re-watering (t2), ten ‘Dusa’ rootstocks per trial and treatment (control, mild-WS, severe-WS) were
inoculated with 3.75 g of colonized wheat seeds per litter of substrate. To ensure
the spread of the inoculum, it was placed in eight points scattered around the stem
(~3.5 cm apart) and introduced at two depths (~5 cm and ~15 cm, respectively). Disease
progression was evaluated by measuring the aerial symptoms of WRR according to a scale:
1= healthy plant; 2= mild wilting; 3= wilting; 4=desiccated; and 5= death. The disease
index (DI) on each treatment and the area under the disease progress curve (AUDPC)
was calculated as previously described by Teixeira de Sousa  and Campbell and
Madden , respectively.
Data were analysed using the analytical software STATISTICA 7 (StatSoft, Inc., USA).
Differences among treatments in physiological variables and AUDPC were evaluated by
analysis of variance (ANOVA). Significant differences were considered at the 5% probability
level unless otherwise stated. Prior to ANOVA, normality and homogeneity assumptions
were tested by using the Kolmogorov–Smirnov and the Cochran's C test, respectively.
When significant differences were found, Fisher’s least significant difference (LSD)
test was used to compare mean values. Statistical analysis of qRT-PCR data was carried
out by Student´s t-test with Sigma Stat version 4.0 software (Systat Software GmbH).