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Plant Pathology. 2024;00:1–12. wileyonlinelibrary.com/journal/ppa
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1© 2024 British Society for Plant Pathology.
1 | INTRODUCTION
Wild rocket (Diplotaxis tenuifolia) belongs to the Brassicaceae fami ly,
which includes important agricultural vegetable crops such as broc-
coli, cabbage, cauliflower and oilseed rape. The general designation
of ‘rocket’ plants refers to Brassicaceae species used to flavour sal-
ads. The most common are D. tenuifolia, also known as wild or pe-
rennial wall- rocket, and Eruca sativa, the cultivated or salad rocket
(Pignone, 1997). The Mediterranean region, particularly the western
part of the basin, is considered the centre of diversity of the genus
Diplotaxis and encompasses around 30 different species (Martínez-
Laborde, 19 96). Despite the similarities between species, mainly in
the initial development stages, dif ferences are recognized between
Diplotaxis and Eruca genera in terms of plant architecture, leaf mor-
phology, chromosomal number and phytochemical compound con-
tent (Tripodi et al., 2017 ). Recently, simple- sequence repeat (SSR)
and single- nucleotide polymorphism- cleaved amplified polymor-
phic sequences (SNP- CAPS) molecular markers were identified and
Received: 24 April 2024
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Accepted: 16 July 2024
DOI : 10.1111/p pa.13 979
ORIGINAL ARTICLE
Histological characterization of downy mildew infection in wild
rocket (Diplotaxis tenuifolia)
Ana L. Pereira1 | Paula Scotti- Campos1,2 | Paula S. Coelho1,3
1INIAV–Institu to Nacional de Investigação
Agrária e Veterinária, Oeiras, Portugal
2GeoBioTec, Faculdade de Ciência s e
Tecnologia, Universidade Nova de Lisboa,
Caparica, Portugal
3GREEN- IT, Universidade Nova de Lisboa
(ITQB/NOVA), Oeiras, Portugal
Correspondence
Paula S. C oelho, INIAV–Instituto Nacional
de Invest igação Agrária e Veterinária,
Avenida da Re pública, 2780- 505 Oeiras,
Portugal.
Email: paula.coelho@iniav.pt
Funding information
REMIRUCULA - Resis tance
characterization to downy mildew
in wild rocket crop, Grant/Award
Number: https://doi.org/10.54499/
UIDB/04551/2020, https://doi.
org/1054499/UIDP/04551/2020 and
PTDC /ASP- PL A/28963/20 17
Abstract
Wild rocket downy mildew (DM), caused by oomycete Hyaloperonospora sp., is a widely
spread disease reducing crop production and quality. New productive wild rocket va-
rieties resistant to DM are crucial to control disease and ensure high quality leaves.
A histological characterization of Hyaloperonospora sp. infection was performed in 11
wild rocket (Diplotaxis tenuifolia) accessions with contrasting DM responses (R, resist-
ant; PR, partially resistant; S, susceptible). Samples of infected cotyledons, first and
second leaves of 14- day- old seedlings were collected at 3, 6, 9, 21 and 24 h post-
inoculation (hpi) and 7 days post- inoculation (dpi) and stained for appressoria and
haustoria observation and tissue necrosis evaluation. Occurrence of appressoria on
host surfaces was higher in leaves compared with cotyledons but unrelated with DM
resistance response. Haustoria growth in mycelium was delayed in R accessions from
3 hpi, and signs of cell hypersensitivity reaction were observed at 9 hpi. At 24 hpi, ne-
crotic spots limited pathogen growth in resistant accessions, whilst mycelium invaded
larger mesophyll areas and produced more haustoria in susceptible ones. At 7 dpi,
sporulation was heavy in S, restricted to droplet deposition sites in PR, and did not
occur in R hosts. A rapid response of R accessions resulted in slower mycelium growth
and longer infection periods. These traits have important agronomic value and should
be considered in germplasm selection for breeding programmes aimed at crop protec-
tion. A better understanding of the host response to DM infection will allow selection
of more suitable wild rocket accessions in future breeding programmes.
KEYWORDS
Brassicaceae, downy mildew resistance, histology, Hyaloperonospora sp., plant–pathogen
interaction, seedling stage
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PEREIRA et al.
allowed an unequivocal discrimination between D. tenuifolia and
E. sativa accessions (Reis et al., 2022).
Rocket vegetables have been used for food, cosmetics and me-
dicinal purposes since ancient times (Cavaiuolo & Ferrante, 2014 ). In
recent years, the consumption of baby leaves (small leaves) has in-
creased remarkably due to their use in ready- to- eat salad packages,
which has resulted in an expansion of farming areas and market trad-
ing in Europe, the United States and Asia (Bell & Wagstaff, 2014).
Wild rocket is often consumed in mixtures with other baby leaves
(e.g., lettuce, spinach, chicory, endive, lambs' lettuce and Swiss
chard). Increased production of these vegetables was triggered by
consumers pursuing a healthier lifestyle and demanding sustainable
and nutritionally rich food products. Rocket leaves contain high lev-
els of fibre a nd important mineral nutr ients, but also ant ioxidant mol-
ecules such as ascorbic acid, flavonoids and carotenoids (Cavaiuolo
& Ferrante, 2014). The presence of glucosinolates and their vola-
tile isothiocyanate derivatives confer to wild rocket a strong bitter
spicy taste and a peculiar highly appreciated pungent aroma (Bell
& Wagstaff, 2019; Hall et al., 2012; Nicoletti et al., 20 07; Tripodi
et al., 2017). It contains bioactive compounds potentially linked to
the prevention of inflammatory diseases and certain types of cancer
(Lucarini et al., 2022; Tripodi et al., 2017).
Intensive production under protected and open field conditions
and the introduction of new highly productive susceptible varieties
increased the occurrence of diseases and pests (Saini et al., 2017).
Wild rocket downy mildew (DM) is a devastating disease responsible
for severe yield losses and quality depreciation (Caruso et al., 2018;
Choi et al., 2018; Nicoletti et al., 2007). This disease is caused by
the oomycete Hyaloperonospora sp. (Oomycota, Peronosporaceae), an
obligate biotrophic pathogen that requires a living host to achieve
its survival and reproduction. The infection in E. sativa is caused by
Hyaloperonospora erucae, but the Hyaloperonospora sp. pathogen
that infects D. tenuifolia does not yet have a described species name
(Choi et al., 2018). Symptoms usually appear as small and irregular
dark brown/chlorotic speckles on infected organs. Mature conidio-
phores (profuse sporulation) become visible a few days later as white
and dense structures mainly on the abaxial side of leaves in compat-
ible host–pathogen interactions. Green leaves infected with DM are
unmarketable (Gullino et al., 2019).
Hyaloperonospora sp. can survive in crop residues in the soil,
overwintering as quiescent sexually produced thick- walled oospores
that in temperate zones are responsible for primary infection under
favourable conditions. The crucial role played by oospores in over-
wintering has been recognized in other downy mildew pathosystems
(Lebeda & Cohen, 2011; Spring et al., 2019; Spring & Zipper, 2000).
In field conditions, the airborne asexual conidia formed in conidio-
phores are the most important means of disease spread throughout
the growing season and cause local infections on leaves within less
than a week (short period of asexual reproduction). The germina-
tion of Hyaloperonospora sp. conidia occurs through the formation
of a germ tube on the plant surface that directly penetrates the
epidermis. Once the epidermis is crossed, the mycelium grows in-
tercellularly and emit s haustoria. These specialized struc tures have
an important function in pathogen feeding and suppression of host
defence by secretion of targeted effectors (Kamoun et al., 2015). In
other plant- pathogenic oomycetes, such as in Plasmopara viticola, the
causal agent of downy mildew disease on grapevine, germination be-
gins through zoospores released from sporangia that penetrate the
plant through the stomata (Kiefer et al., 2002).
Chemical control in wild rocket crops is problematic due to the
increasingly restrictive regulations of pesticide applications, the
short crop cycle and multiple harvests, therefore requiring the use of
plant protection products with reduced preharvest inter vals (Caruso
et al., 2018). Sustainable strategies to control DM disease and en-
sure high production and quality are urgently needed and include
the combined use of resistant cultivars and alternative control meth-
ods, as well as agronomic practices that reduce foliar humidity. New
varieties must be appealing to consumers but also resistant to the
main biotic and abiotic stress factors (Gullino et al., 2019). Several
collections of brassica germplasm have been screened for resistance
to DM and different sources of resistance were identified (Coelho
et al., 1998; Coelho & Monteiro, 2018; Coelho, Reis, et al., 2022;
Coelho, Valério, & Monteiro, 2022).
Histological examination of infected organs provides evidence of
pathogen biology and host plant defence responses that cannot be
distinguished macroscopically. Development of oomycete infection
structures and colonization of plant tissues contribute to identifica-
tion of host resistance mechanisms, such as the plant hypersensi-
tivity response based on programmed cell death. This study aimed
to characterize at the histological level 11 D. tenuifolia genotypes
differing in their responses to DM infection (resistant, partially resis-
tant and susceptible). A better understanding of the host responses
to DM infection will allow the selection of more suitable wild rocket
accessions for future breeding programmes.
2 | MATERIALS AND METHODS
2.1 | Plant material and growth conditions
A set of 11 D. tenuifolia populations and commercial varieties
with different DM responses (six resistant, R; two partially resist-
ant, PR; and three susceptible, S) were selected from a collection
of wild rocket germplasm belonging to the Instituto Nacional de
Investigação Agrária e Veterinária, IP, Portugal (Table 1). These ac-
cessions were previously screened for DM resistance, and disease
indexes (DI) calculated at the seedling stage under controlled condi-
tions (Coelho, Reis, et al., 2022) and at the adult stage in the field
(Coelho et al., 2023). These results enabled the selection of the ac-
cessions used in this study.
The 11 accessions were sown in the same tray (8 × 11 cells) in
a total of 12 trays, corresponding to the six incubation times and
two treatments (control vs. inoculated). A peat- based compost
(Gramoflor GmbH & Co. KG) was used and seeds were covered with
vermiculite and watered by a capillary watering system. The trays
were placed in a growth chamber at 21°C day/19°C night , 70%–80%
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PEREIRA e t al.
relative humidity (RH) and a 19- h light photoperiod at a light inten-
sity of 250 μmo l/m2/s (LED ECOT814330F 14 W 4000 K; Roblan) for
14 days until seedling inoculation.
2.2 | Inoculation methodology
The Hyaloperonospora sp. isolate D5 was obtained from an infected
commercial wild rocket (variety Athena T Z 1441) produced under
organic farming conditions in 2019 (Odemira, Portugal). After path-
ogen isolation, the inoculum was stored at −18°C for later use in
screening tests.
The 11 accessions were infected with a fresh conidial suspen-
sion of Hyaloperonospora sp. pathogen multiplied in the susceptible
wild rocket accession S10 (Table 1), and the concentration of spores
was adjusted to 50–75 × 103 conidia/mL (Coelho, Reis, et al., 2022).
Cotyledons, first and second leaves (leaf 1 and leaf 2, respectively)
of 14- day- old seedlings were individually inoculated with two 10 μL
droplets of the conidial suspension, in a total of 48 plants per acces-
sion (8 plant s × 6 incubation times). Control plants were inoculated
with sterile distilled water. After inoculation, plantlets were incubated
in the dark (16°C, 90 ± 5% RH) for 24 h to stimulate Hyaloperonospora
sp. infection. During this period, sampling was performed at 3, 6, 9,
21 and 24 h post- inoculation (hpi). Then, the final group of plants was
transferred to the grow th chamber with the initial growth conditions
for 5 days. Af ter a 24- h dark period, before the last sampling for his-
tological observation at 7 days post- inoculation (dpi), plants were
screened for DM resistance through visual observation to confirm
the DI evaluation from previous studies (Coelho, Reis, et al., 2022).
2.3 | Staining procedure for microscopic
observation
In each accession and incubation time, the cotyledons, first and
second leaves were collected individually and immersed separately
in different tubes with 96% ethanol to allow decolouration then
stored at 4°C. A total of 16 cotyledons and eight first and eight sec-
ond leaves were prepared per treatment. For microscopic obser-
vation, the samples were double stained with aniline blue- trypan
blue (Nowicki et al., 2012) to detect the different structures of the
pathogen along with the plant host defence responses. Briefly, the
samples stored in ethanol were rehydrated by soaking in decreas-
ing ethanol solutions (75% and 50%). Subsequently, they were im-
mersed in an aqueous solution of 0.05% tr ypan blue overnight at
room temperature, followed by an immersion in 0.05% aniline blue
in 150 mM KH2PO4 solution (pH 9) for 3–4 h. After that period, the
samples were washed for 15 min in the same solution (two or three
times) and mounted in water for microscopic observation.
A total of 45 germinated conidia (15 conidia × 3 biological repli-
cates) were individually scored in each accession, organ and incuba-
tion time. The presence or absence (1/0) of appressoria (expressed
as a percentage) and the number of haustoria were quantified using
a bright- field microscope (Wetzlar Dialux 20; Leica) coupled to a
Accession
code Name
Seed
donoraStatus
Cotyledon/leaves/adult plantb
Disease index DM reaction
R1 Eros TZ5622 Tozer
Seeds
Cultivar 2.0/1.6/0.4 R/R/R
R2 Tel es to R Z8 9- 00 5 Rijk Zwaan Cultivar 1.8/1.6/0.6 R/R/R
R3 Poseidon TZ5623 Tozer
Seeds
Cultivar 1.7/1.6/0.9 R /R/R
R4 DIP LO14 IPK Selfing line 1.0/1.0/0.0 R/R/R
R5 94818CRU C2 AGG Selfing line 1.9/1.1/0.0 R/R/R
R6 Unknown Unknown Selfing line 2. 5/1 .7/0.1 R/R/R
PR7 Zeus TZ 5505 Tozer
Seeds
Cultivar 3.0/1.8/2 .3 PR/R/PR
PR8 84329 Unknown Unknown 3.3/2.3/2.5 PR/R/PR
S9 DIPLO12 IPK Selfing line 5.0/5.7/n.t. S/HS/n.t.
S10 Unknown Unknown Selfing line 6.0 /4.6 /3.7 HS/S/HS
S11 948 31CRU C2 AGG Selfing line 6.0/5.8/4 .1 HS/HS/HS
aSeed companies: Tozer Seeds and Rijk Zwaan. Genebank collections: Leibniz Institute (IPK) and
Australian Grains Genebank (AGG).
bAccording to the disease index (DI) the accessions were separated into four phenot ypic
categories. Seedling stage (cotyledon and first two leaves): R, resistant (DI ≤2.5); PR, partially
resistant (2.5 < DI ≤4.0); S, susceptible (4.0 < DI ≤5.0); and HS, highly susceptible (5.0 < DI ≤6.0)
(Coelho, Reis et al., 2022). Adult plant stage: R, resistant (DI ≤1.0); PR, partially resistant (1.0 < DI
≤2.5); S, susceptible (2.5 < DI ≤3.5); and HS, highly susceptible (3.5 < DI ≤5.0) (Coelho et al., 2023).
DM response of accessions: R1 to R6, resist ant; PR7 and PR8 , partially resistant; and S9 to S11,
susceptible.
TABLE 1 List of Diplotaxis tenuifolia
accessions tested at seedling (cotyledons,
1st and 2nd leaves) and adult plant stages,
under controlled and field conditions,
respectively, and their reaction to
Hyaloperonospora sp. infection expressed
as disease index.
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PEREIRA et al.
Moticam 1800 camera (Motic Europe). The presence of necrosis in
host tissues was also evaluated throughout the assay.
2.4 | Statistical analysis
Data are presented as mean values and standard errors of 45 co-
nidia observed in cotyledons and first and second leaves. The forma-
tion of appressoria and the number of haustoria were analysed by
analysis of variance (ANOVA) using STATISTIC A v. 12.0. Means were
compared using Tukey HSD test (α = 0.05). Correlation coefficients
of Pearson were calculated to compare appressoria and haustoria
growth between cotyledons and leaves.
3 | RESULTS
3.1 | Appressoria formation
The presence or absence of appressoria within the 24 hpi period sig-
nificantly differed among D. tenuifolia accessions. Resistant acces-
sion R5 showed the lowest value (75%) and the susceptible S9 the
highest (89%) (Figure 1).
In general, the incidence of appressoria in cotyledons was lower
(80%) compared to leaves 1 (85%) and leaves 2 (84%), which were
not significantly different from each other. Cotyledons presented
the highest variation, with values ranging from 68% (accession R5)
to 94% (accession S9). The variation was smaller in leaves 1, between
75% (R5) and 91% (R6). In relation to leaves 2 (the youngest leaves),
no significant differences occurred between accessions, varying be-
tween 81% (R1) and 89% (PR8) (Table 2). Five distinct accessions (R3,
R4, R6, PR7 and S11) did not show dif ferences between cotyledons
and the two leaves. In accessions R1, R2, R5, PR8 and S10, the coty-
ledons presented lower appressoria values compared to one or both
leaves. Only in the susceptible accession S9 did the cotyledons show
a significantly higher value (94%) than leaves 2 (85%) (Figure 2).
Regarding the five incubation times (Table 2), a lower percentage
of appressoria was obser ved in all organs at 9 hpi, suggesting that
an external factor was responsible for the decrease at this incuba-
tion time. The appressoria in cotyledons varied between 63% (9 hpi)
and 88% (3 hpi). In first leaves, excluding the 9 hpi (65%), the other
incubation times did not vary significantly, recording mean values
between 87% (6 hpi) and 92% (21 hpi). In these leaves, at 3 hpi and
6 hpi no differences were found in appressoria among accessions,
and at 21 hpi only the resistant R5 registered a lower value (51%)
compared to the remaining accessions. Regarding second leaves, the
mean value of appressoria varied between 62% (9 hpi) and 94% (3
hpi) (Table 2).
No significant correlations were observed regarding the for-
mation of appressoria among the three organs during the 24 hpi
period (Figure S1): cotyledons and first leaves (r = 0.575 ns, n = 11);
cotyledons and second leaves (r = 0.170 ns, n = 11); first leaves and
second leaves (r = 0.488 ns, n = 11). Furthermore, there was no rela-
tionship between appressorium formation and the DM response of
the accessions.
3.2 | Haustoria formation
The number of haustoria observed in Hyaloperonospora sp. myce-
lium was significantly different among accessions within the 24 hpi
period. In general, in resistant accessions the number of haustoria
was lower than in susceptible ones. The resistant accessions R1, R2
and R3 had the lowest values (0.7, 0.8 and 0.8, respectively), while
the susceptible S10 and S11 showed a higher number (1.7 and 1.8,
respectively). In the 24 hpi period, the haustoria number in resistant
R6 was significantly higher (1.3) compared to partially resistant PR7
and resistant R4 accessions, which presented the same value as each
other (1.1) (Figure 3).
In the same period, dif ferences were observed among organs,
with leaves 1 showing the highest value (1.3), followed by leaves 2
(1.2) and cotyledons (1.1) (Table 3). However, four accessions (R6,
PR8, S9 and S10) did not show significant differences among organs.
In accessions R1, R2, R3, R4, R5 and PR7, leaves 1 presented a higher
number compared to cotyledons (Figure 4). The number of haustoria
in different accessions did not differ between the t wo leaves, except
FIGURE 1 Appressoria formation
(mean ± SE ) in Hyaloperonospora sp.
conidia on 11 Diplotaxis tenuifolia
accessions. Accession means and SE were
calculated from 675 observations (15
observations × 3 biological replicates × 3
organs × 5 incubation times). Means were
compared by the Tukey test and different
letters indicate significant differences
(α = 0.05). Downy mildew response of
accessions: R1 to R6, resistant; PR7 and
PR8, partially resistant; and S9 to S11,
susceptible.
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PEREIRA e t al.
in PR7 and S11, in which first leaves presented higher values than
second leaves. These accessions had a slower growth and second
leaves were still expanding.
Regarding the formation of haustoria at dif ferent incubation
times (Table 3), pathogen infection was delayed in resistant acces-
sions (except in R6 accession) compared to partially resistant and
TABLE 2 Percentage of appressoria (mean ± SE) in cotyledons, first and second leaves of 11 Diplotaxis tenuifolia accessions infected by
Hyaloperonospora sp.
Organ Accession
Incubation time (hours post- inoculation)
Mean3 6 9 21 24
Cotyledons R1 86.7 ± 5 a 82.2 ± 6 ab 60.0 ± 7 bc 82.2 ± 6 ab 66.7 ± 7 cd 75. 6 bcd
R2 62.2 ± 7 b 80.0 ± 6 ab 5 7.8 ± 7 bc 77. 8 ± 6 ab 91.1 ± 4 abc 73.8 cd
R3 80.0 ± 6 ab 88.9 ± 5 ab 80.0 ± 6 ab 82.2 ± 6 ab 76.6 ± 6 abcd 81.3 bc
R4 88.9 ± 5 a 93.3 ± 4 ab 66.7 ± 7 ab 64.4 ± 7 b 95. 6 ± 3 a 81.8 bc
R5 84.4 ± 5 a 91.1 ± 4 ab 33.3 ± 7 c 37. 8 ± 7 c 93.3 ± 4 ab 68.0 d
R6 86.7 ± 5 a 95.6 ± 3 a 53.3 ± 8 bc 100.0 ± 0 a 91.1 ± 4 abc 85.3 ab
PR7 95.6 ± 3 a 84.4 ± 5 ab 93.3 ± 4 a 93.3 ± 4 a 64.4 ± 7 d 86.2 ab
PR8 95.6 ± 3 a 73.3 ± 7 b 53.3 ± 8 bc 66.7 ± 7 b 91.1 ± 4 abc 76. 0 bc d
S9 97. 8 ± 2 a 9 7.8 ± 2 a 84.4 ± 5 ab 97. 8 ± 2 a 93.3 ± 4 ab 94. 2 a
S10 97. 8 ± 2 a 82.2 ± 6 ab 5 7.8 ± 7 bc 93.3 ± 4 a 62 .2 ± 7 d 78.7 bcd
S11 93.3 ± 4 a 95.6 ± 3 a 53. 3 ± 8 bc 97. 8 ± 2 a 68.9 ± 7 bcd 81.8 bc
Mean 88.1 A 87.7 A 63.0 C 81.2 B 81.2 B 80.2 b
Leaf 1 R1 95.6 ± 3 a 77. 8 ± 6 a 71.1 ± 7 ab 93.3 ± 4 a 88.9 ± 5 ab 85.3 ab
R2 80.0 ± 6 a 91.1 ± 4 a 53.3 ± 8 abc 97. 8 ± 2 a 100.0 ± 0 a 84.4 abc
R3 84.4 ± 5 a 80.0 ± 6 a 57.8 ± 7 abc 97. 8 ± 2 a 100.0 ± 0 a 84.0 abc
R4 88.9 ± 5 a 84.4 ± 5 a 35.6 ± 7 c 88.9 ± 5 a 100.0 ± 0 a 79. 6 bc
R5 86.7 ± 5 a 86.7 ± 5 a 48.9 ± 8 bc 51 .1 ± 8 b 100.0 ± 0 a 74. 7 c
R6 95.6 ± 3 a 88.9 ± 6 a 82.2 ± 6 a 100.0 ± 0 a 88.9 ± 5 ab 91.1 a
PR7 86.7 ± 5 a 86.7 ± 5 a 73.3 ± 7 ab 100.0 ± 0 a 7 7.8 ± 6 b 84.9 ab
PR8 91.1 ± 4 a 82. 2 ± 6 a 73.3 ± 7 ab 88.9 ± 5 a 95.6 ± 3 ab 86.2 ab
S9 91.1 ± 4 a 93.3 ± 4 a 71.1 ± 7 ab 100.0 ± 0 a 84.4 ± 5 ab 88.0 ab
S10 95.6 ± 3 a 91.1 ± 4 a 73.3 ± 7 ab 100.0 ± 0 a 82.2 ± 6 ab 88.4 ab
S11 95.6 ± 3 a 95.6 ± 3 a 75 .6 ± 6 ab 100.0 ± 0 a 7 7.8 ± 6 b 88 .9 ab
Mean 90.1 A 87.1 A 65.1 B 92.5 A 90.5 A 85.1 a
Leaf 2 R1 97. 8 ± 2 a 82.2 ± 6 ab 64.4 ± 7 ab 80.0 ± 6 b 82.2 ± 6 ab 81.3 a
R2 75.6 ± 6 b 93.3 ± 4 ab 46.7 ± 8 b 100.0 ± 0 a 100.0 ± 0 a 8 3.1 a
R3 97. 8 ± 2 a 93.3 ± 4 ab 5 7.8 ± 7 b 95.6 ± 3 ab 86.7 ± 5 ab 86. 2 a
R4 93.3 ± 4 a 88.9 ± 5 ab 42.2 ± 7 b 86.7 ± 5 ab 100.0 ± 0 a 82.2 a
R5 97. 8 ± 2 a 86.7 ± 5 ab 73.3 ± 7 ab 51 .1 ± 8 c 100.0 ± 0 a 81.8 a
R6 95.6 ± 3 a 73.3 ± 7 b 60.0 ± 7 b 95.6 ± 3 ab 97. 8 ± 2 ab 84.4 a
PR7 91.1 ± 4 ab 88.9 ± 5 ab 5 7.8 ± 7 b 97. 8 ± 2 ab 82. 2 ± 6 ab 83.6 a
PR8 91.1 ± 4 ab 73.3 ± 7 b 93.3 ± 4 a 95.6 ± 3 ab 93.3 ± 4 ab 89.3 a
S9 93.3 ± 4 a 95.6 ± 3 a 64.4 ± 7 ab 91.1 ± 4 ab 80.0 ± 6 ab 8 4.9 a
S10 97. 8 ± 2 a 93.3 ± 4 ab 62 .2 ± 7 ab 100.0 ± 0 a 84.4 ± 5 ab 87.6 a
S11 97. 8 ± 2 a 95.6 ± 3 a 55. 6 ± 7 b 95. 6 ± 3 ab 7 7. 8 ± 6 b 84.4 a
Mean 93.5 A 8 7.7 B 61.6 C 8 9.9 A B 89. 5 AB 84.4 a
Mean 90.6 A 87. 5 A 63.2 B 87. 8 A 87. 1 A
Note: Accession means and standard errors were calculated from 45 observations (15 observations × 3 biological replicates) for each organ and incubation
time. Values followed by different letters are significantly different according to the Tukey test (α = 0.05). Lowercase letters (in column) refer to the
comparison of the accessions within each incubation time, and uppercase letters (in line) refer to the comparison among incubation times (A/a letters
refer to the highest values). Downy mildew response of accessions: R1 to R6, resistant; PR7 and PR8, partially resistant; and S9 to S11, susceptible.
6
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PEREIRA et al.
susceptible ones. At 3 hpi, the cotyledons, first and second leaves
of resistant accessions (R1, R2, R4 and R5) were not yet showing
haustoria. The absence of haustoria indicates that Hyaloperonospora
penetration was unsuccessful at this incubation time in resistant
accessions, except for accession R3 which recorded a mean value
of 0.04 haustoria in leaves 2, and the accession R6 (haustoria in all
organs). Haustoria formation was recorded at 3 hpi in all par tially
resistant and susceptible accessions.
At 3 and 6 hpi, no visible microscopic signs of necrosis were ob-
served in mesophyll cells invaded by Hyaloperonospora sp. pathogen
or in their vicinity (Figure 5a–c). At 9 hpi, tissue necrosis started to
be visible in resistant accessions (Figure 5d), in contrast to obser-
vations in partially resistant (Figure 5e) and susceptible (Figure 5f)
wild rocket.
At 21 hpi, mycelium growth and haustoria formation began to
differ among D. tenuifolia accessions according to DM susceptibility
(Figure 5g–h). In resistant (R1 to R5) and partially resistant (PR7)
accessions, the invading mycelium and haustorium formation pro-
gressed very slowly. The number of haustoria varied bet ween 0.3 (R4)
and 2.7 (PR8) in cotyledons, between 0.8 (R1) and 3.1 (S10) in leaves
1, and between 0.6 (R2) and 2.8 (PR8 and S10) in leaves 2 (Table 3).
At 24 hpi, tissue colonization in the three infec ted organs in-
creased significantly, especially in compatible interactions. The num-
ber of haustoria varied from 0.9 (R1) to 3.9 (S11) in cotyledons, from
1.5 (R3) to 3.7 (S11) in leaves 1, an d from 1.0 (R3) to 3.2 (S10) in leaves
2 (Table 3). At the same time, limitation of mycelium growth due to
necrotic spots was evident in incompatible interactions (Figure 5i). In
contrast, in susceptible accessions, the Hyaloperonospora sp. patho-
gen had already invaded a larger area of the mesophyll and exhibited
an increase in the number of haustoria (Figure 5k). At 7 dpi, success-
ful infections culminated with the reproduction of the pathogen, as
observed in accession S11 (Figure 5p).
FIGURE 2 Appressoria formation (mean ± SE) in Hyaloperonospora sp. conidia on the infected sur face of cotyledons, first and second
leaves of 11 Diplotaxis tenuifolia accessions. Accession means and standard errors were calculated from 225 observations (15 observations
× 3 biological replicates × 5 incubation times) for each organ. The organs of each accession were compared by the Tukey test and different
letters indicate significant differences (α = 0.05). Downy mildew response of accessions: R1 to R6, resistant; PR7 and PR8, partially resistant;
and S9 to S11, susceptible. ns, not significant.
FIGURE 3 Number of haustoria (mean ± SE) in Hyaloperonospora sp. mycelium in 11 Diplotaxis tenuifolia accessions. Accession means and
standard errors were calculated from 675 observations (15 observations × 3 biological replicates × 3 organs × 5 incubation times). Means
were compared by the Tukey test and dif ferent letters indicate significant differences (α = 0.05). Downy mildew response of accessions: R1
to R6, resist ant; PR7 and PR8, partially resistant; and S9 to S11, susceptible.
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PEREIRA e t al.
Significant correlations were found in the number of haustoria
of cotyledons, first and second leaves during the 24 hpi period. The
highest correlation coefficient was observed between cotyledons
and first leaves (r = 0.983***, n = 11), followed by first leaves and sec-
ond leaves (r = 0.825***, n = 11), and cotyledons and second leaves
(r = 0.778**, n = 11) (Figure 6). This result indicates that cotyledons
TABLE 3 Number of haustoria (mean ± SE ) in cotyledons, first and second leaves of 11 Diplotaxis tenuifolia accessions infected by
Hyaloperonospora sp.
Organ Accession
Incubation time (hours post- inoculation)
Mean3 6 9 21 24
Cotyledons R1 0.00 ± 0.00 d 0.56 ± 0.12 a 0 .51 ± 0.11 de 0.38 ± 0.12 e 0.91 ± 0.18 e 0.47 f
R2 0.00 ± 0.00 d 0.69 ± 0.12 a 0.62 ± 0.12 de 0.44 ± 0.12 e 1.09 ± 0.22 de 0.57 ef
R3 0.00 ± 0.00 d 0.58 ± 0.10 a 0.49 ± 0.12 e 0.67 ± 0.15 de 1.33 ± 0.22 de 0.61 ef
R4 0.00 ± 0.00 d 1.02 ± 0.13 a 1.00 ± 0.13 cde 0.33 ± 0.10 e 1.78 ± 0. 22 cde 0.83 def
R5 0.00 ± 0.00 d 0.73 ± 0.14 a 1.56 ± 0.16 bc 1.36 ± 0.16 cd 1.13 ± 0.17 de 0.96 d
R6 0.07 ± 0.04 cd 0.64 ± 0.12 a 0.84 ± 0.15 de 2.22 ± 0.18 ab 1.82 ± 0.24 cde 1.12 c d
PR7 0.49 ± 0.0 8 a 0.60 ± 0.12 a 1.13 ± 0.15 bcd 0. 89 ± 0.17 de 2.02 ± 0.24 cd 1.03 cd
PR8 0.40 ± 0.07 ab 0.89 ± 0.12 a 0.84 ± 0.13 de 2.71 ± 0.25 a 1.73 ± 0.23 cde 1.32 bc
S9 0.22 ± 0.06 bcd 0.89 ± 0.11 a 1.71 ± 0.16 ab 1.82 ± 0.19 bc 2.47 ± 0.21 bc 1.42 bc
S10 0.27 ± 0.07 abc 0 .91 ± 0.12 a 0.89 ± 0.14 de 2.53 ± 0.17 ab 3.07 ± 0.23 ab 1.53 b
S11 0.40 ± 0.07 ab 0.96 ± 0.13 a 2.07 ± 0.17 a 2.53 ± 0.17 ab 3 .93 ± 0.19 a 1.98 a
Mean 0.17 E 0.77 D 1.06 C 1.44 B 1.94 A 1.08 c
Leaf 1 R1 0.00 ± 0.00 d 0.53 ± 0.10 bcd 1.31 ± 0.15 b 0.78 ± 0.13 e 1.58 ± 0.19 d 0.84 g
R2 0.00 ± 0.00 d 0.29 ± 0.08 cd 1.51 ± 0.16 ab 1.09 ± 0.17 de 2. 24 ± 0.20 cd 1.03 efg
R3 0.00 ± 0.00 d 0.22 ± 0.07 d 1.0 4 ± 0.15 b 1 .96 ± 0.19 bc 1.47 ± 0.19 d 0.94 fg
R4 0.00 ± 0.00 d 0.58 ± 0.10 bcd 1.31 ± 0.14 b 1.29 ± 0.15 cde 2.69 ± 0.18 bc 1.17 de f
R5 0.00 ± 0.00 d 0.53 ± 0.11 bcd 2.07 ± 0.16 a 1.49 ± 0.14 cde 2.38 ± 0.17 cd 1.29 cde
R6 0.09 ± 0.04 cd 0.53 ± 0.12 bcd 1.04 ± 0.14 b 1.67 ± 0.15 cd 3.31 ± 0.19 ab 1.33 cd
PR7 0.29 ± 0.07 bc 0.71 ± 0.12 abc 1.33 ± 0.13 b 2.47 ± 0.17 ab 1.6 4 ± 0.23 d 1.29 cde
PR8 0.18 ± 0.06 bcd 0.36 ± 0.09 cd 1.53 ± 0.18 ab 2.60 ± 0.17 ab 2.64 ± 0.19 bc 1.46 bcd
S9 0.27 ± 0.07 bc 0.44 ± 0.08 cd 1.73 ± 0.14 ab 2.42 ± 0.18 ab 2.78 ± 0.29 bc 1.53 bcd
S10 0.56 ± 0.07 a 1.07 ± 0.12 a 1.67 ± 0.18 ab 3.07 ± 0.17 a 2.33 ± 0.21 cd 1.74 ab
S11 0.33 ± 0.07 ab 0.96 ± 0.11 ab 1.64 ± 0.16 ab 2.6 4 ± 0.15 ab 3.71 ± 0.19 a 1.86 a
Mean 0.16 E 0.57 D 1.47 C 1.95 B 2.4 3 A 1.32 a
Leaf 2 R1 0.00 ± 0.00 b 0.47 ± 0.09 ab 1.82 ± 0.19 a 0.71 ± 0.14 c 1.22 ± 0.19 c 0.84 c
R2 0.00 ± 0.00 b 0.31 ± 0.09 b 1.49 ± 0.16 abcd 0.56 ± 0.13 c 1.76 ± 0. 21 bc 0.82 c
R3 0.04 ± 0.03 b 0.44 ± 0.10 b 1.49 ± 0.18 abcd 1.89 ± 0.19 b 1.02 ± 0.18 c 0.98 c
R4 0.00 ± 0.00 b 0.36 ± 0.09 b 1.18 ± 0.12 abcd 1.98 ± 0.18 b 2.89 ± 0.17 a 1.28 b
R5 0.00 ± 0.00 b 0.42 ± 0.09 b 1.56 ± 0.13 abc 1.93 ± 0.18 b 2.82 ± 0.17 a 1.35 b
R6 0.11 ± 0.05 b 0.4 4 ± 0.11 b 0.87 ± 0.11 d 2.36 ± 0.17 ab 2 .91 ± 0.18 a 1.34 b
PR7 0.13 ± 0.05 b 0. 51 ± 0.10 ab 0.96 ± 0.13 cd 1.16 ± 0.14 c 1.87 ± 0.19 bc 0.92 c
PR8 0.20 ± 0.06 b 0.44 ± 0.07 b 1.13 ± 0.15 bcd 2. 82 ± 0.18 a 2.98 ± 0.15 a 1.52 ab
S9 0.64 ± 0.07 a 0.47 ± 0.10 ab 1.22 ± 0.13 abcd 2.09 ± 0.14 b 2.62 ± 0.24 ab 1. 41 b
S10 0.56 ± 0.07 a 0.49 ± 0.09 ab 1.64 ± 0.15 ab 2.82 ± 0.11 a 3. 24 ± 0.22 a 1.75 a
S11 0.13 ± 0.05 b 0.89 ± 0.11 a 1.29 ± 0.14 abcd 2.18 ± 0.13 ab 2 .58 ± 0.18 ab 1.41 b
Mean 0.17 E 0.48 D 1.33 C 1.86 B 2.36 A 1 .24 b
Mean 0.16 E 0.60 D 1.29 C 1 .76 B 2.24 A
Note: Accession means and standard errors were calculated from 45 observations (15 observations × 3 biological replicates) for each organ and incubation
time. Values followed by different letters are significantly different according to the Tukey test (α = 0.05). Lowercase letters (in column) refer to the
comparison of the accessions within each incubation time, and uppercase letters (in row) refer to the comparison among incubation times (A/a letters
refer to the highest values). Downy mildew response of accessions: R1 to R6, resistant; PR7 and PR8, partially resistant; and S9 to S11, susceptible.
8
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PEREIRA et al.
and first leaves are the most appropriate organs to test DM response
in 14- day- old D. tenuifolia plantlets.
The progression of Hyaloperonospora sp. infection was affected
by the resistance response of D. tenuifolia accessions from early
stages of infection (24 hpi period) and became more pronounced
throughout the colonization period. A clear separation was observed
among accessions according to their response to DM. The group of
resistant accessions had the lowest number of haustoria, the par-
tially resistant presented an intermediate value and the susceptible
group recorded the highest number (Table 3).
3.3 | Phenotypic expression of resistance
Macroscopic differences were also evident among accessions.
Necrosis in mesophyll cells were visible in all accessions and turned
to a brownish colour due to the Hyaloperonospora sp. mycelium and
haustoria growth in different periods.
At 7 dpi, in resistant accessions, strong necrotic spots were ob-
served macroscopically in the inoculation points in cotyledons and
leaves (Figure 5l). These accessions did not show conidiophores;
however, in some plants very few conidiophores (up to five) devel-
oped occasionally, restricted to the infection drops and protruding
from the stomatal pore (Figure 5m). In partially resistant accessions,
at 24 hpi necrotic spots were microscopically visible at the inocu-
lation site in some cotyledons and leaves (Figure 5j). At 7 dpi, the
mycelium grew between mesophyll cells with the formation of haus-
toria surrounded by necrotic tissue (Figure 5n).
At 7 dpi, in susceptible accessions necrotic areas were visible to
the naked eye but did not prevent the growth of Hyaloperonospora
sp. mycelium and haustoria. Hyaloperonospora sp. pathogen com-
pleted its life cycle by forming conidiophores bearing conidia on
the abaxial side of the cotyledon and leaves. These structures were
widespread all over the mesophyll and massive emergence of co-
nidiophores with conidia could be observed protruding from the
stomata on the abaxial side of susceptible cotyledons and leaves
(Figure 5o,p).
4 | DISCUSSION
Wild rocket genotypes expressing seedling and field resistance to
DM were previously identified and characterized by visual observa-
tion at cotyledon and adult plant stages (Coelho et al., 2023; Coelho,
Reis, et al., 2022). The latter study revealed that the DM resistance
observed at the seedling stage in wild rocket is a good indicator of
the resistance behaviour expressed by adult plants in the field.
In the present study, the response of D. tenuifolia seedlings to
Hyaloperonospora sp. infection was examined at the histological level
in an attempt to clarify the processes responsible for restricting
pathogen invasion. Hyaloperonospora sp. infection begins with the
germination of conidia in water drops deposited on leaves. The germ
tube grows until it reaches a ridge on the host surface and forms a
simple anchoring appressorium at the tip, facilitating the infec tion
process, although there are cases in which the infectious process
occurs without the formation of an appressorium.
At 3 hpi the germination of Hyaloperonospora sp. conidia had
already occurred over epidermal cells in all wild rocket accessions.
Between 3 and 24 hpi, a high percentage of conidia (average above
80%) formed an appressorium in cotyledons (80%), first leaves (85%)
and second leaves (84%). The percentage of appressoria observed
in the two leaves did not vary significantly. No clear relationship
was obser ved between the appressorium formation and the DM
response of the accessions, as also repor ted for Bremia lactucae (let-
tuce downy mildew) by Lebeda et al. (2001, 2006).
However, in this work, higher values tended to occur in leaves
rather than in cotyledons. In some DM, the siting of appresso-
ria appears to be highly specific, suggesting an interaction with
host surface topography or chemistry (Lucas et al., 1995). Lebeda
and Reinink (1991) reported significant differences in incidence of
FIGURE 4 Number of haustoria (mean ± SE) in Hyaloperonospora sp. mycelium in infected cotyledons, first and second leaves of 11
Diplotaxis tenuifolia accessions. Accession means and standard errors were calculated from 225 observations (15 observations × 3 biological
replicates × 5 incubation times) for each organ. The organs of each accession were compared by the Tukey test in each accession and
different letters indicate significant differences (α = 0.05). Downy mildew response of accessions: R1 to R6, resist ant; PR7 and PR8, partially
resistant; and S9 to S11, susceptible. ns, not significant.
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PEREIRA e t al.
appressorium formation between cotyledons (higher frequency) and
leaf discs of adult plants of lettuce cultivars 6 h after inoculation.
Regarding the five incubation times, a lower percentage of ap-
pressoria was observed in all organs at 9 hpi, suggesting that an
external factor was responsible for the decrease at this incubation
time. Dif ferent hypotheses may be considered, such as variations
in relative humidity conditions in the plant chamber at this time.
Another possibility is that, despite inoculum tubes being kept in ice,
9 hpi plants were the last to be inoculated and had a longer period
between inoculum preparation and plant inoculation, which may
have affected appressoria formation. However, it was not possible
to verify these hypotheses in this assay.
DM infection in wild rocket is similar to other brassicas, such
as broccoli, oilseed rape and mustard; however, in radish and non-
cruciferous crops (e.g., pea, lupin and wheat) the appressorium is
absent or scarce (Li et al., 2 011). The present results indicate that
the appressorium formation does not depend on the resistant or
susceptible response of the wild rocket accession. Therefore, this
cannot be considered as a good parameter to differentiate ac-
cessions concerning their DM response and does not seem to be
a crucial step in infection and colonization success in wild rocket.
Furthermore, Li et al. (2011) suggested that the initial progression
of the pathogen infection may be primarily determined by other fac-
tors apar t from specific signals from plants. Too little is known about
FIGURE 5 Microscopic observation of the oomycete Hyaloperonospora sp. pathogen infection in cotyledons of wild rocket (Diplotaxis
tenuifolia) accessions with different responses to downy mildew at different incubation times; R1 to R6, resistant; PR7 and PR8, partially
resistant; and S9 to S11, susceptible. (a) Conidia germination on the surface of accession R5 at 3 hours post- inoculation (hpi). (b) Conidia
germination on the surface of accession S11 at 3 hpi. The germ tube grows until it reaches a ridge of cotyledon/leaves and forms an
appressorium in the tip. (c) Mycelium and haustoria growth in accession S11 at 6 hpi. (d) Mycelium and haustoria growth in accession R1 at 9
hpi. (e) Mycelium and haustoria growth in accession PR8 at 9 hpi. (f) Mycelium and haustoria growth in accession S11 at 9 hpi. (g) Mycelium
and haustoria growth in accession R1 at 21 hpi. (h) Mycelium and haustoria growth in accession S11 at 21 hpi. (i) Mycelium with necrotic
spots at the inoculation site in accession R1 at 24 hpi. (j) Mycelium with haustoria formation and necrosis onset in accession PR8 at 24
hpi. (k) Mycelium with haustoria growth without necrosis in accession S11 at 24 hpi. (l) Necrotic areas limit mycelium growth and prevent
Hyaloperonospora sp. sporulation in accession R5 at 7 days post- inoculation (dpi). (m) Two Hyaloperonospora sp. conidiophores protruding
from the stomatal pore in the cotyledon of accession R1 at 7 dpi. (n) Limited mycelium growth and necrotic areas did not prevent haustoria
formation in accession PR8 at 7 dpi. (o) Extensive mycelial growth with haustoria invading cotyledon of accession S11 at 7 dpi. (p) Massive
emergence of conidiophores with tip ellipsoid conidia formation in cotyledon abaxial surface of accession S11 at 7 dpi. Bar in a, b, c, e, f, h,
k = 20 μm; bar in g, i, j, m, n = 25 μm; bar in d, l, o, p = 50 μm.
10
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PEREIRA et al.
the factors triggering appressorium formation in downy mildews
(Lebeda et al., 2001). As the infection progresses, the mycelium in-
vades the mesophyll through intercellular spaces emitting haustoria
and expands by invaginating the cell membrane and the cytoplasm,
but never penetrates the host plant cell wall. This structure is sur-
rounded by the extrahaustorial membrane, a continuum of the plant
plasma membrane with a unique composition (Koh et al., 2005).
At 3 hpi, no haustoria were observed in the resistant acces-
sions R1, R2, R4 and R5, while in partially resistant and susceptible
wild rocket accessions, a first haustorium immediately developed
on the epidermal or mesophyll cells. Regarding the two other re-
sistant accessions, R3 presented some haustoria in leaf 2, and R6
in cotyledon, first and second leaves. The accession R6, previously
classified as resistant (DI = 2.5 at the cotyledon stage; Coelho,
Reis, et al., 2022), showed a similar response to partially resistant
accessions PR7 and PR8 (3.0 and 3.3, respectively), reflecting some
degree of susceptibility. This result suggests that material classi-
fied with DI = 2.5 at the cotyledon stage can be considered par-
tially resistant. The plants used in the previous evaluation (Coelho,
Reis, et al., 2022) and in this study, despite coming from the same
set of seeds, were derived from plant populations, thus presenting
some natural variability.
Infection hyphae penetrate directly between the anticlinal cell
walls of two adjacent epidermal cells both in cotyledons and leaves.
The delay observed in Hyaloperonospora sp. haustoria formation into
the epidermis of wild rocket resistant accessions (R1 to R5) com-
pared to partially and susceptible accessions has also been obser ved
in other interactions and crops (Li et al., 2011; Zhu et al., 2022). In
studies conducted on host and non- host crops infected by H. par-
asitica, Li et al. (2011) reported that the first haustorium is formed
in the epidermal cell adjacent to the penetrating point at 2 hpi in all
brassicas, except in B. carinata, B. juncea and Raphanus raphanistrum,
which developed haustoria later (8 hpi). In Lupinus angustifolius and
Trifolium subterraneum the penetration may occur either directly or
through stomata (Li et al., 2011).
The time Hyaloperonospora sp. takes to colonize the host tissue
varies among accessions. At 24 hpi in resistant genotypes, a few
haustoria denoted the expansion of the mycelium through the me-
sophyll cells, while susceptible accessions presented more hausto-
ria and enhanced mycelium growth. A longer infection period and
limited sporulation are of great agronomic interest in crop protec-
tion. Lebeda et al. (2006) observed that the frequency of haustorial
formation varies specifically among Lactuca spp. genotypes carrying
different Dm genes and/or R- factors for host resistance. In compati-
ble host–parasite interactions, the frequency and size of haustoria is
significantly higher than in incompatible interactions.
In this study greater leaf maturity seems to be a key factor for in-
fection, with leaves 1 presenting a higher average haustoria number
than younger leaves 2, especially in accessions PR7 and S11. This is
in agreement with obser vations in other brassicas, confirming that
leaf age influences DM infec tion, so that it is more difficult for juve-
nile leaves that are not fully expanded to become infected (Agnola
et al., 2003; Coelho et al., 2009). In slower growing accessions (e.g.,
PR7 and S11), leaves 2 are young and still expanding, resulting in a
delay in the establishment of infection despite susceptibility of the
accessions. Infection formation can be influenced by organ surface,
namely the number and character of trichomes, thickness and com-
position of waxes and number and position of stomata, among oth-
ers, which may determine the success or failure of pathogen spore
deposition and subsequent ingress by infection structures (Lebeda
et al., 2001).
Resistance genes in plants can activate the hypersensitivity
response (HR), a signal transduction pathway leading to a lo-
calized programmed cell death that prevents further pathogen
spread and a disease resistance response (Yaeno et al., 2004).
HR is considered to be a major step that prevents further devel-
opment of biotrophic pathogens (Lebeda et al., 2001). The HR in
Brassica spp. infected by fungal and oomycete pathogens includes
various responses, such as cell necrosis close to arrested hyphae,
phytoalexin, callose and lignin secretion, aggregation of pectin
FIGURE 6 Significant correlations determined by Pearson coef ficient were observed between the number of haustoria developed on
cotyledons, first and second leaves in 11 Diplotaxis tenuifolia accessions. (a) Correlation between cotyledons versus first leaves (r = 0.983,
***p ≤ 0.001, n = 11). (b) Correlation between cotyledons versus second leaves (r = 0.778, **p ≤ 0.01, n = 11). (c) Correlation between first
versus second leaves (r = 0.825, ***p ≤ 0.001, n = 11). Accession means were calculated from 225 observations (15 observations × 3 biological
replicates × 5 incubation times) for each organ. Downy mildew response of accessions: R1 to R6, resistant; PR7 and PR8, partially resistant;
and S9 to S11, susceptible.
(a) (b) (c)
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11
PEREIRA e t al.
in the lumen of xylem vessels and induction of pathogenesis-
related (PR) proteins including β- 1,3- glucanase and chitinase
(Mourou et al., 2023). HR is a particularly efficient response
against biotrophic pathogens such as mildew diseases that de-
pend on host cell nutrients for their development and reproduc-
tion (Michelmore et al., 1988). In this study, the beginning of a
typical HR of cells was perceived through microscopy observa-
tion at 9 hpi in resistant accessions, in contrast to that observed
in partially resistant and susceptible ones. This is in accordance
with the observations reported by Li et al. (2011). Tissue necrosis
was also responsible for DM resistance in radish accessions with
the inhibition and reduction of H. brassicae growth within 24 hpi
(Coelho & Monteiro, 2018).
A macroscopic inspection of plants at 3 dpi showed a slight
necrosis/chlorosis in resistant accessions, clearly seen at the inoc-
ulation (drop deposition) sites. At 5 dpi, the susceptible accessions
showed the first visual signs of sporulation. At 7 dpi, all accessions
showed visible necrosis, although to a dif ferent extent. The final
phase of successful tissue colonization culminated with the growth
of the reproductive structures in compatible interactions.
At 7 dpi, no sporulation occurred in resistant accessions (with the
exception of very few plants). In partially resistant accessions, the
sporulation in cotyledons and leaves was restricted to the droplet
deposition site. In susceptible hosts, very abundant sporulation was
observed, with hyphae protruding from stomata and forming conidio-
phores that developed conidia on the tips of the branch, especially on
the abaxial leaf surface. In severe infections a whitish mould appeared
on both leaf surfaces. Leaves suffered a sudden yellowing and pre-
mature defoliation occurred. Cotyledons in D. tenuifolia are small and
age quickly. In 14- day- old wild rocket plants, cotyledons and leaves 1
seem to be more reliable than leaves 2 in evaluation of DM response.
The present study contributes to a better understanding of
response mechanisms underlying interactions between the re-
sistant and susceptible wild rocket accessions and the pathogen
Hyaloperonospora sp. Results demonstrate that the resistant ac-
cessions presented a delay in host infection and a reduced patho-
gen growth associated with an HR (necrotic spots) that prevented
or strongly limited pathogen reproduction. These results highlight
agronomic traits of utmost importance for crop protection that
contribute to the development of more suitable genotypes through
breeding programmes. Future research should be undertaken to un-
ravel the resistance mechanisms underlying this wild rocket–Hyalo-
peronospora sp. pathosystem and to evaluate pathogen population
diversity to identify the existence of pathotypes/races.
ACKNOWLEDGEMENTS
This work was supported by the project PTDC/ASP- PLA /28963/2017
and ALG- 01- 0145- FEDER- 028963 (REMIRUCULA—Resistance
characterization to downy mildew in wild rocket crop) and the FCT—
Fundação para a Ciência e a Tecnologia, IP, through the R&D Unit
‘GREEN- IT–Bioresources for Sustainability’ UIDB/04551/2020
(https:// doi. org/ 10. 54499/ UIDB/ 04551/ 2020) and UIDP/04551/2020
(https:// doi. org/ 10. 54499/ UIDP/ 04551/ 2020).
DATA AVAIL ABILI TY STATEMENT
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
ORCID
Paula S. Coelho https://orcid.org/0000-0002-7133-998X
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SUPPORTING INFORMATION
Additional supporting information can be found online in the
Suppor ting Information section at the end of this article.
How to cite this article: Pereira, A.L ., Scotti- C ampos, P. &
Coelho, P.S. (2024) Histological characterization of downy
mildew infection in wild rocket (Diplotaxis tenuifolia). Plant
Pathology, 00, 1–12. Available from: ht tps://doi.o rg/10.1111/
ppa.13979
