Leaf position-dependent effect of Alternaria brassicicola development on host cell death,

photosynthesis and secondary metabolites in Brassica juncea

Violetta K. Macioszeka,*, Marzena Wielanekb, Iwona Morkunasc, Iwona Ciereszkoa and Andrzej
K. Kononowiczd
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a
Department of Plant Physiology, Faculty of Biology and Chemistry, University of Bialystok, 15-245
Bialystok, Poland
b
Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection,
University of Lodz, 90-237 Lodz, Poland
c
Department of Plant Physiology, Poznan University of Life Sciences, 60-637 Poznan, Poland
d
Department of Plant Ecophysiology, Faculty of Biology and Environmental Protection, University of
Lodz, 90-237 Lodz, Poland

Correspondence
*Corresponding author,
e-mail: v.macioszek@uwb.edu.pl

During the first 24 hours of infection, Alternaria brassicicola developmental parameters such as
conidial germination, germ tubes and appressoria formation on each of the five mature Brassica
juncea leaves, correlated with a leaf position showing stronger development of the pathogen on older
leaves than on young ones. As a consequence of fungal development, the black spot disease was
observed during 96 hours of infection on a macroscopic scale, as well as via confocal microscopy.
Degradation of the chloroplast thylakoids and plastoglobule appearance during infection, followed by
the decrease in chlorophyll a fluorescence parameters i.e. maximum quantum yield of PSII (Fv/Fm),
non-photochemical quenching (NPQ) and chlorophyll a:b ratio, have been observed. Also, after an
initial increase of carbohydrates (glucose, fructose and sucrose), content far below the respective
control values was found. The content of secondary metabolites such as flavonoids and glucosinolates
increased in a leaf position-dependent manner in infected leaves, with a lower level in older leaves
than in younger ones. Although, the total phenolic compounds (TPCs) content did not differ
significantly in infected leaves compared to control leaves, TPCs level in both control and infected
leaves was leaf position-dependent. To the best of our knowledge, this is the first report on leaf
position-dependent effect on the B. juncea biochemical response to A. brassicicola infection.

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 Abbreviations – chl a:chl b, chlorophyll a:b ratio; Fv/Fm, maximum quantum yield of PSII; hpi, hours
post-inoculation; NPQ, non-photochemical quenching; PSII, photosystem II; TPCs, total phenolic
compounds.

Introduction
Indian mustard (Brassica juncea (L.) Czern.) is an important crop cultivated mainly in Eastern Europe,
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India, China, North America and Australia for its seeds and leaves. The seeds are used as a spice and a
source of edible oils, as well as in the production of mustard, while the fresh leaves can be an addition
to salads and also feed for farm animals (Warwick 2010, Rahman et al. 2018). Moreover, B. juncea
can be used in the phytoremediation of soil and water contaminated by metals (Podar et al. 2004,
Singh and Fulekar 2012, Rizwan et al. 2018). Recently, cultivars of Indian mustard have been
investigated thoroughly because of their potential therapeutic effect on cancer, cardiovascular and
neurodegenerative diseases (Björkman et al. 2011, Kwak et al. 2016, Frazie et al. 2017).
The necrotrophic fungus Alternaria brassicicola causes the black spot disease in all Brassica species
worldwide and is among the most destructive Brassica fungal pathogens such as Alternaria brassicae,
Leptosphaeria maculans and Sclerotinia sclerotiorum (Lawrence et al. 2008, Nowicki et al. 2012).
Most species of cultivated Brassicas, including Indian mustard, show various degrees of susceptibility
to A. brassicicola, although there is no known fully resistant cultivar. In general, Alternaria species
produce host-specific and non-host specific toxins that manifest different modes of action on plant
cells. Toxins can affect cell division, protein synthesis, membrane permeabilization, chloroplast photo-
phosphorylation and also target ER, nucleus, vacuole and Golgi bodies (Otani et al. 1995, Thomma
2003, Meena et al. 2017). However, the A. brassicicola main virulence factor is not known to date.
Recently, it has been determined that major metabolites produced by A. brassicicola during Brassica
plant infection are phytotoxin brassicicolin A, depudicin and siderophore N,N-dimethylcoproge
(Pedras et al. 2009, Pedras and Park 2015). On the other hand, Brassicas foliar tissues contain many
secondary metabolites such as glucosinolates and a broad spectrum of phenolic compounds that show
antimicrobial activity, but such defence is insufficient against A. brassicicola due to the detoxification
mechanism (Pedras and Hossain 2011, Srivastava et al. 2013).
Successful infection of a susceptible host by a necrotrophic fungus such as A. brassicicola depends on
many environmental factors, mainly temperature and humidity, as well as on the host plant age and
conidial concentration (Dixon 1981, Deep and Sharma 2012). Host plant age seems to play a crucial
role in the development of black spot disease symptoms - the older the plants, the more severe the
disease symptoms that have been observed during infection of various B. rapa and B. oleracea
cultivars at seedling and mature leaf stages (Doullah et al. 2006, Nowakowska et al. 2019). However,
screening of 160 B. napus cultivars for resistance against A. brassicicola at the cotyledon and mature
plant stages led to the observation of different cultivars susceptible to the infection at the cotyledon

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 stage rather than those susceptible at the mature plant stage (Macioszek et al., unpublished data). This
phenomenon is probably related to the differences in metabolite content at various developmental
stages of the plant that can affect the infection (Meena et al. 2011). It should be emphasized that the
physiological state of particular leaves of mature Brassica plants also influences the black spot disease
severity. Infected true leaves attached at the 1st or 2nd position (the older one) show more severe
symptoms than at the 3rd or 4th position (Doullah et al. 2006). However, research on leaf position-
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dependent progression of disease in various pathosystems is rarely accompanied by biochemical
and/or physiological analyses of a host.
Here we report on the leaf position-dependent influence in the susceptible B. juncea cultivar on A.
brassicicola development and its impact on the progress of the disease in a particular mature leaf using
a macroscopical as well as a light and electron microscopical analysis. We also focus on changes in
the host primary metabolism, such as a photosynthetic apparatus efficiency during the infection of
mature leaves, as well as in primary and secondary metabolites production such as carbohydrates,
glucosinolates and phenolic compounds during infection.

Materials and methods

Plant and fungal material

Indian mustard (Brassica juncea (L.) Czern.) seeds were sown in soil and grown to a stage of five
mature leaves (about 4 weeks) in a plant growth room under controlled conditions: 22°C, 60% relative
humidity and light intensity 120 μmol m-2 s-1 (Super TLD Philips 865), and a 16 h day/8 h night
photoperiod. Fungus Alternaria brassicicola (ATCC 96836) was grown on potato dextrose agar plates
(Difco) in the same conditions as the plants, but in the dark. Each of the five leaves was inoculated
with two or four 10 µl drops of conidial suspension at a concentration 5x105 conidia per ml or for
control plants with drops of distilled water. The plants were then incubated in transparent boxes to
maintain high humidity.

Fungal development
Samples were harvested cutting 1.2 cm discs of the infected leaf area with a cork borer at 4, 6, 8, 10,
12, 14, 16, 20, and 24 hpi (hours post-inoculation) from three plants per time point. The leaf discs
were boiled in 70% ethanol to remove chlorophyll and stained with 0.5% aniline blue in lactophenol
(w/v) and mounted in lactophenol. Random images from the inoculation area were captured under a
light microscope (Olympus BX) and the germinating conidia, germ tubes and appressoria per 100
conidia were counted for each disc. The germ tubes were counted when their length was at least half
the length of a conidium.

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 Plant disease assay
The inoculated plants were analysed for the visible spread of necrosis every 24 h for 4 days.
Measurement of the necrosis diameter was performed with a calliper. Six plants per experiment were
inoculated. Each experiment was repeated three times.

Confocal laser scanning microscopy (CLSM)

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The leaves of control and infected plants were scanned using a Leica TCS SP8 confocal laser scanning
microscope and Leica TCS LSI confocal stereo laser scanning microscope - equipped with a
monochromatic Leica camera at 24, 48, 72 and 96 hpi. Images were captured under a long path filter
with an excitation wavelength of 488 nm and emission spectra of 500-770 nm and merged using Leica
LAS-AF software version 3.3.0. All images were subjected to alterations changing red to magenta
using Photoshop v. 6.0 according to the Publisher's protocol for colour vision impaired readers.

Transmission electron microscopy (TEM)

For the ultrastructural investigation of chloroplasts, samples (0.3 x 0.3 mm) of control and inoculated
leaves after 24 and 48 hpi were used. They were fixed in 3% glutaraldehyde in 0.1 M cacodylate
buffer, pH 6.8, at 0-4°C for 8 h. After washing in cacodylate buffer and post-fixation in 2% osmium
tetroxide for 3 h, the samples were dehydrated in an ethanol series, subsequently infiltrated with the
mixture of Epon-Spurr resin and propylene oxide and embedded in Epon-Spurr resin. The ultra-thin
sections (80 nm) were obtained using a Reichert Jung microtome (Leica). Ultra-thin sections, after
staining with uranyl acetate and lead citrate, were examined with TEM (JEOL 1010) at 80 kV.

Chlorophyll analysis
Chlorophyll was extracted in 100% methanol and estimated according to Wellburn (1994). The ratio
of chlorophyll a:b was counted for three control and three inoculated plants per experiment. The
experiment was repeated four times (n = 4).

Chlorophyll a fluorescence measurements

The photosynthetic parameters, like the photochemical efficiency of photosystem II, were measured
using the Handy FluorCam 1000-H system (Photon Systems Instruments) under an actinic light at 150
µmol (photons) m-² s-1. The control and infected plants were dark-adapted for 30 min and the
maximum quantum yield of photosystem II (Fv/Fm) was measured. In the light-adapted state, the non-
photochemical quenching (NPQ) was estimated. Six control and six infected plants were used per
experiment. The experiment was repeated three times (n = 3).

Carbohydrate analysis

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 The plant material (150 mg) was ground in liquid nitrogen and extracted with 80% methanol. The
samples were supplemented with 25 µl ribitol (1 mg ml-1), mixed in a thermomixer for 10 min at room
temperature and centrifuged at 11 000g for 10 min at 4°C. The supernatant was evaporated in a speed
vac at room temperature. After sample desiccation, each sample was supplemented with 50 µl
methoxyamine (20 mg ml-1 in dry pyridine) and mixed for 1.5 h at 37°C. Following short spin
centrifugation, the samples were supplemented with 80 µl MSTFA (Sigma-Aldrich), mixed again (30
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min at 37°C) and centrifuged at 11 000 g for 10 min. Endogenous carbohydrate levels were
determined by gas chromatography coupled with mass spectrometry (GC-MS; 6890N gas
chromatograph by Agilent with GCT Premier mass spectrometer by Waters) using a DB-5MS column
(30 m x 0.25 mm x 0.25 µm, J&W Scientific). For separation of volatile compounds, a temperature
gradient was used as follows: 70°C for 2 min, followed by 10°C per min, up to 300°C (10 min). For
sample injection, a PTV injector was used in the range of 60-250°C, interface 250°C, the transfer line
temperature was set to 250°C and source to 250°C. Spectra were recorded in m/z range of 50-650 in
EI+ mode with an electron energy of 70 eV. For quantitative analysis, calibration curves for glucose,
fructose and sucrose were prepared in the range of 10-150 µg per sample. Carbohydrate content was
expressed in mg g-1 fresh weight. Three control and three infected plants were used per experiment and
the experiment was repeated twice. The means were calculated from six measurements (n = 6).

Secondary metabolite analyses

For metabolite analysis, approximately 100 mg of plant material per sample from control and infected
plants at 24, 48, 72 and 96 hpi were harvested, frozen in liquid nitrogen and stored at -70°C until used.
The content of metabolites was determined using a PowerWave XS microplate spectrophotometer
(BioTek) against a blank prepared with concentrated methanol, as for the plant extract preparation for
the respective assays. Three control and three infected plants were used in each experiment.
Plant samples were extracted in 80% methanol HPLC grade (POCh) and the same extracts were used
for total phenolic compounds and flavonoid analyses.
The content of total phenolic compounds (TPCs) was determined spectrophotometrically at 725 nm
according to the standard Folin-Ciocalteu method (Ainsworth and Gillespie 2007, Blainski et al.
2013). The TPCs content was expressed as mg g-1 fresh weight based on the calibration curve for
gallic acid (Sigma-Aldrich). The experiment was repeated three times (n = 3).
Flavonoid analysis was performed by the aluminium chloride colorimetric method (Pękal and
Pyrzynska 2014). The flavonoid content was determined spectrophotometrically at 415 nm and
expressed as mg g-1 fresh weight based on the calibration curve for quercetin (Sigma-Aldrich). The
experiment was repeated three times (n = 3).
Glucosinolates were extracted with boiling 70% (v/v) methanol and purified according to the
previously published protocol (Wielanek and Urbanek 2006). Briefly, the glucosinolate extract was

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 purified in alumina columns containing acidic aluminium oxide and eluted with 1% K2SO4 (w/v) using
the SPE (solid phase extraction) system. To remove excess sulphate, the eluents were combined with
ethanol (1:1, v/v) and centrifuged (34 000 g). The supernatant was used to analyse the total
glucosinolate fraction according to the modified version of the DeClercq and Daun (1989) method
with anthrone instead of thymol. The concentration of the resulting colour complex of glucosinolate-
glucose with anthrone was determined spectrophotometrically at 620 nm. Glucosinolate content was
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expressed as mg g-1 fresh weight based on the calibration curve for the sinigrin standard (Sigma-
Aldrich). The experiment was repeated twice (n = 2). The means were calculated from six
measurements (n = 6).

Statistical analysis
Statistical analysis was performed with ANOVA and Duncan's test using STATISTICA v. 12.5.
All the figures presented in this paper were composed using Photoshop v. 6.0.

Results

Quantitative fungal development

The number of germinating conidia, germ tubes and appressoria of Alternaria brassicicola were
counted on five mature leaves of Brassica juncea plants from the oldest leaf (1st one) to the youngest
one (5th one) during the first 20 hours of infection (Fig. 1, Appendix S1). Conidia started germinating
about 2-3 hpi (hours post-inoculation) on the oldest leaf (data not shown) and about 4 hpi on the
youngest one. Approximately 80% of conidia germinated during the first 10 hpi on the 1st and 2nd
leaves, whereas 40 and 18% of conidia germinated on the 4th and 5th leaves, respectively. Over 85%
of germinating conidia on the 1st leaf could be observed from 12 hpi and their number increased to
95% at 20 hpi. The number of germinating conidia gradually increased on each leaf at each time point,
attaining 90% on the 2nd leaf, over 85% on the 3rd leaf and over 80% on 4th and 5th leaves at 20 hpi.
The number of germ tubes on the 1st and 2nd leaves increased significantly over the number of
germinating conidia at 12 and 16 hpi, respectively. The number of germ tubes and germinating conidia
on the 3rd, 4th and 5th leaves was equal from 16 hpi at the following time points. First appressoria on
the 1st, 2nd and 3rd leaves were observed at 6 hpi, but on the 4th and 5th leaves at 8 hpi. Germinating
conidia, germ tubes and appressoria manifested a weak negative correlation with leaf position (P <
0.001), but a high positive correlation between A. brassicicola development parameters and post-
inoculation time was discovered (P < 0.001, Appendix S1). The germ tubes and appressoria
appearance on leaves was strongly correlated with germinating conidia (P < 0.001). Germinating
conidia formed a mycelial network on the 1st and 2nd leaves at 24 hpi, whereas on the 5th leaf germ

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 tubes and hyphae displayed less density and were shorter (Appendix S1). The first brownish necrotic
cells on the 1st and 2nd leaves were observed at 14 hpi and on the 3rd, 4th and 5th leaves at 16 hpi.

Disease development
The spread of necrotic lesions was observed on each of the five mature leaves of B. juncea plants
infected with A. brassicicola conidial suspension during 96 hpi (Fig. 2). Visible necroses appeared at
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the inoculation site at 24 hpi and they spread over the inoculation area at 48 hpi. Chlorotic rings
around the necroses were visible at 72 and 96 hpi. In case of most observed 1st leaves, the chlorotic,
dead area covered the whole leaf at 96 hpi (Fig. 2C). Positive correlation between necrosis diameter
and post-inoculation time (r = 0.80, r2 = 0.64, P < 0.001) was observed in case of each leaf
investigated (Fig. 2D), but a weak negative correlation was found between necrosis diameter and leaf
position (r = -0.37, r2 = 0.14, P = 0.0032). However, significant differences in the necrosis diameter
between all leaf positions at 96 hpi were observed according to the Duncan`s test (P < 0.05).

Cell death
Infected leaves without any pre-treatment were observed under a confocal microscope at 24, 48, 72
and 96 hpi. Control leaves with a drop of water demonstrated magenta autofluorescence with a
constant intensity that indicated chlorophyll fluorescence and only a weak green autofluorescence
background (Appendix S2). Dark holes at the inoculation sites in magenta autofluorescence images at
24 hpi correlated with the areas of necrosis development, which, on the other hand, showed strong,
intense green autofluorescence (Appendix S2). 3D images of necrotic areas on the 1st and 3rd leaves
at 48 hpi showed an uneven spreading area of necrosis and less dense leaf tissue structure than on the
5th leaf, whereas the necrosis border on the 5th leaf was clearly separated from the surrounding
healthy tissue (Fig. 3). At 48 and 72 hpi (Appendix S2), it was found that the older the leaf, the greater
the necrosis and the stronger the green autofluorescence. There was large chlorosis around the necrotic
area and a strong green autofluorescence on the 1st and 2nd leaves and almost no chlorophyll
autofluorescence at 96 hpi (Fig. 4). Green autofluorescence on the 3rd, 4th and 5th leaves at analysed
time points was strictly correlated with the necrotic area.

Chlorophyll a fluorescence analysis

Formation of necrosis on B. juncea leaves started with the formation of chlorosis (and decrease of
chlorophylls content, data not shown) which indicated probable changes in photosynthetic metabolism
and chloroplast structure. Significant leaf position-dependent differences in the ratio of chlorophyll
a:b in control leaves could be observed, although there was no correlation between chlorophyll a:b
ratio and post-inoculation time (Fig. 5A, Appendix S3). However, chlorophyll a:b ratio in infected
leaves manifested a significant decrease compared to the control values according to Duncan's test (P

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 < 0.05) and was dependent on both leaf position and post-inoculation time (Appendix S3). In the case
of the Fv/Fm parameter in control plants, a similar trend as for the ratio of chlorophyll a:b was found
(Fig. 5B). Fv/Fm values were stable during the investigated time points in control leaves, but increased
in younger leaves from 0.78 in the 1st leaf to 0.86 in the 5th leaf. In infected leaves, the values of
Fv/Fm featured a significant decrease correlated with both post-inoculation time and leaf position
(Appendix S3). However, the differences in Fv/Fm values between control and infected leaves
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decreased in younger leaves, attaining no significant difference for the 5th leaf. Similar trends as for
the Fv/Fm parameter were observed for NPQ values in control and infected plants (Fig. 5C, Appendix
S3), but NPQ values in an infected 5th leaf showed a significant decrease compared to the control at
48 and 72 hpi (P < 0.05).

Changes in chloroplast structure

Ultrastructural investigation revealed that hyphae grew into the apoplast of the 1st and 3rd leaves at 24
hpi (Figs 6B and B') and caused a disturbance in the chloroplast structure, revealing large starch grains
and thylakoids degeneration (Figs 6C and 6C'), compared to the chloroplasts in control leaves Figs 6A
and 6A'). Further changes at 48 hpi in the chloroplast structure of the 1st and 3rd leaves included the
degeneration of outer and inner chloroplast membranes, the disintegration of thylakoids and the
appearance of plastoglobules (Figs 6D and 6D').

Carbohydrate content
Production of large starch grains in chloroplasts of B. juncea leaves during infection with A.
brassicicola suggested possible changes in sugar content, fluxes or distribution. GC-MS analysis of
glucose, fructose and sucrose revealed significant differences in their contents in control and infected
leaves. Glucose content in both the 1st and 3rd control leaves did not change significantly at the
investigated time points, but glucose content in infected leaves featured a leaf position-dependent
negative correlation (Appendix S3) and it was significantly higher in the 1st infected leaf compared to
the 3rd (Fig. 7A). Fructose content in control and infected leaves did not change significantly during
post-inoculation time but was negatively correlated with leaf position (Fig. 7B, Appendix S3). It has to
be emphasized that both glucose and fructose content was significantly higher in the 1st infected leaf
compared to the control leaf at investigated time points, but glucose and fructose levels in the 3rd
infected leaf were significantly below the control values at 72 hpi. Sucrose content increased in control
leaves in a time-dependent manner, but it decreased in infected leaves below the control values at 72
hpi after the initial increase at 24 hpi (Fig. 7C, Appendix S3). The level of sucrose was weakly
correlated with leaf position in infected plants (Appendix S3).

Phenolics and glucosinolate content

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 There was no significant difference in the total phenolic compounds (TPCs) content between the
control and infected plants (Fig. 8A), but TPCs content depended on leaf position (Appendix S3).
However, it has to be emphasized that TPCs content in infected leaves showed a trend over its content
in the respective control leaves. Flavonoids content was highly correlated with leaf position both in
control and infected plants (Appendix S3). Although flavonoid content differed significantly between
control and infected plants in older leaves (P < 0.05) with a higher content in infected leaves, but
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mostly it did not depend on post-inoculation time (Fig. 8B, Appendix S3). Glucosinolate content also
revealed significant differences between the control and infected plants (P < 0.05) with a higher
content in infected plants (Fig. 8C). The content of glucosinolates was strongly correlated with leaf
position in both control and infected plants, but it also featured a weak positive correlation with post-
inoculation time in infected leaves (Appendix S3).

Discussion
In recent years, several papers have reported on Brassica juncea response to Alternaria brassicicola
infection focusing mainly on the host phytohormone response (Mazumder et al. 2013, Meur et al.
2015). However, albeit of significant importance, the B. juncea-A. brassicicola pathosytem is rather
poorly characterised in regard to the biochemistry of host and infection dynamics. The fact is that most
scientific work focuses on the interaction between B. juncea and another Alternaria species - A.
brassicae. This research is focused on how host leaf position affects both A. brassicicola development
and the B. juncea biochemical response to the infection.

Alternaria brassicicola develops in a leaf position-dependent manner

The infection cycle of A. brassicicola as a plant pathogen is quite simple and it can be accomplished
from a single conidium to conidiogenesis during 24-48 h (Macioszek et al. 2018). The infection
dynamics, aside from environmental conditions also depend on the host - particularly its metabolite
content and its age. It is well-proven that older Brassica plants show more severe disease symptoms
than younger ones during infection by A. brassicicola (Doullah et al. 2006, Deep and Sharma 2012,
Nowakowska et al. 2019). In this study, leaf position-dependent development of A. brassicicola during
B. juncea infection was observed (Fig. 1, Appendix S1). The consequences of this effect could be
observed in necrosis spreading – larger in older leaves compared to young ones (Fig. 2). Such a trend
that upper, younger leaves are more resistant to infection in other Brassica pathosystems has been
observed before (Hong and Fitt 1995, Doullah et al. 2006, Coelho at al. 2009). Doullah et al. (2006)
surprisingly observed more severe disease symptoms on the 5th and 6th true leaves than on the 3rd
and 4th, albeit only in one of many examined B. rapa cultivars susceptible to A. brassicicola infection.

Black spot disease influences Brassica juncea primary metabolism

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 In general, plant infection by a necrotrophic pathogen can affect the primary metabolism of a host
because of the release of phytopathogenic toxins and the host`s tissue damage (Biemelt and
Sonnewald 2006, Berger et al. 2007). It has been observed that in B. juncea leaves infected by A.
brassicicola, chloroplasts could be seriously damaged and, as a consequence, a gradual leaf position-
dependent decrease in the chlorophyll a:b ratio was observed as well as in the basic photosynthetic
parameters such as the Fv/Fm and NPQ (Fig. 6, Appendix S3). The fungal metabolite responsible for
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the chloroplast damage can be tenuazonic acid, a non-host specific phytotoxin produced by many
Alternaria sp., but mainly by Alternaria alternata. Tenuazonic acid is known as a photosynthesis
inhibitor causing chloroplast-derived reactive oxygen species (ROS) generation, chlorophyll
breakdown and a serious damage of chloroplast structure (Chen et al. 2010). It has also been isolated
from A. brassicicola and A. raphani cultures (Gallardo et al. 2004). A decreasing chlorophyll a:b ratio
correlating with infection progress has also been observed during the B. juncea-A. brassicae
susceptible interaction. Higher chlorophyll content in resistant B. alba also has decreased during the
progress of A. brassicae infection (Mathpal et al. 2011). The accumulation of large starch grains in
chloroplasts, also observed during B. juncea infection, was probably another factor that negatively
influenced photosynthetic efficiency (Saharan et al. 2016). Non-invasive chlorophyll fluorescence
measurements are now commonly applied to monitor plant-pathogen interaction effects on
photochemical activity of photosystem II in vivo (Rolfe and Scholes 2010). However, this is the first
report on primary metabolism defects during the B. juncea-A. brassicicola interaction. It has to be
emphasized that although biotic stresses globally down-regulate photosynthesis genes in other
Brassicaceous plants – non-host Arabidopsis thaliana - A. brassicicola infection does not affect this
plant`s photosynthesis (Bilgin et al. 2010). As suspected, changes in photosynthesis efficiency and
chloroplast structure in B. juncea leaves after infection with A. brassicicola influenced carbohydrate
levels in a leaf position-dependent manner (Appendix S3). This is probably related to the preferred
type of carbohydrates as an energy source by A. brassicicola (Cho 2015). Furthermore, an increase in
the activity of invertases, which are enzymes hydrolysing sucrose, is often observed during infection
(Kanwar and Jha 2018). In addition, lower infection dynamics are often observed in younger leaves
during infection with a necrotrophic pathogen (Coelho et al. 2009). The initial increase in fructose,
glucose and sucrose levels in infected B. juncea leaves could also be a result of the defence
mechanism that failed and therefore a progress of the black spot disease with low carbohydrate levels
was observed (Fig. 7). It is believed that depletion of carbohydrates enhances plant susceptibility to a
pathogen infecton (Morkunas and Ratajczak 2014, Bezrutczyk et al. 2018, Kanwar and Jha, 2018). A
decrease in the content of total sugars and reducing sugars in B. juncea during progressive A.
brassicae infection (Mathpal et al. 2011) and Albugo candida infection (Mishra et al. 2009) was
reported.

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 Phenolics level is still maintained during A. brassicicola infection
Plants in the Brassicaceae family are abundant in phenolic compounds, which feature antioxidant and
antimicrobial properties and are considered one of the most important biochemical parameters in
disease resistance (Lattanzio et al. 2006, Cheynier et al. 2013). Indian mustard leaves contain a high
level of free polyphenol, hydroxycinnamic acids and flavonoids (Cartea et al. 2011). Although in this
study the total phenolic compounds (TPCs) content did not significantly differ between control and
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infected leaves, the TPC level was still leaf position-dependent (Fig. 8A, Appendix S3). The results
indicate that despite the spread of necrosis and possible detoxification by A. brassicicola, TPCs
content in all infected leaves is still maintained during progression of the disease to the same extent as
in the control leaves. However, it has been reported that TPCs content gradually increased in B. juncea
leaves infected by A. brassicae (Parihar 2012, Prakash et al. 2012).

Green autofluorescence during infection is probably related to flavonoid synthesis

Plant cell autofluorescence is well-described, mainly because of fluorescence of chlorophyll, which
emits strong red fluorescence under UV light, although many endogenous molecules - mainly phenolic
compounds - in plant cells are fluorophores as well (Talamond et al. 2015). Spectral image analysis
helps to track the progress of disease and abundance of metabolites in the attached leaves (Bellow et
al. 2012). The green autofluorescence restricted to the necrotic and chlorotic area can possibly indicate
the presence of flavonoids according to Buschmann et al. (2000). Flavonoids are polyphenolic
secondary metabolites and are considered to be effective scavengers of ROS and antifungal agents in
plants (Agati et al. 2012). In this study, flavonoid content was strictly correlated with leaf position,
both in control and infected leaves and significantly increased during the infection in lower leaves. In
younger upper leaves (4th and 5th), the flavonoid level was stable and did not differ compared with
the control leaves (Fig. 8B). This effect can be achieved either by the younger leaves reaching the
maximum capacity of flavonoid synthesis, or a successful resistant response against A. brassicicola
infection expressed by the restricted necrotic lesions. However, the decrease in flavonoid content in B.
juncea during progressive infection of A. brassicae has been reported (Parihar 2012, Prakash et al,
2012) and confirmed by the experiments on much older B. juncea plants in the field (Mathpal et al.
2011), but without a leaf position-dependent analysis. The differences of the B. juncea response in
relation to the flavonoid content can be associated with the different mode of action of toxins secreted
by A. brassicicola and A. brassicae during an infection.

Glucosinolate level does not inhibit black spot disease in older leaves
Glucosinolates are one of the most characteristic groups of secondary metabolites in Brassica species
which display antibacterial and antifungal properties in vitro (Sotelo et al. 2015) and contribute to a
resistance to a wide-range of Brassica pathogens including A. brassicicola (Giamoustaris and Mithen

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 1997, Saharan et al. 2016). Although a significantly higher content of glucosinolates in infected B.
juncea leaves was observed when compared with the control (Fig. 8C), progression of disease even in
the upper, younger leaves was undeniable. Similar results for the leaf position-dependent glucosinolate
content and disease development in B. napus leaves during A. brassicae infection were found before
(Doughty et al. 1991). The increased levels of glucosinolates in white cabbage cultivars (B. oleracea)
during artificial inoculation with A. brassicicola in field experiments were also observed (Novotny et
Accepted Article
al. 2018). Synthesis of glucosinolates, although extreme in susceptible Brassica species, was probably
balanced by their detoxification by Alternaria pathogens (Pedras and Abdoli 2017).
In conclusion, research on a response of susceptible cultivars to a pathogen is as important as disease
resistance and it can be useful for establishing disease management strategies. The physiological
response of B. juncea to A. brassicicola infection is highly complex and concerns leaf position-
dependent changes in primary and secondary metabolites as well as photosynthetic parameters and
structural chloroplast alterations. This study indicates a possible mode of action of A. brassicicola
through chloroplasts in Brassica leaves. Furthermore, this paper is the first to demonstrate the
influence of A. brassicicola infection on chloroplast metabolism. The novelty of this work relies on a
comprehensive experimental approach to study the Brassica juncea – Alternaria brassicicola
interaction.

Author contributions
V.K.M. designed and performed the experiments. M.W. performed the glucosinolate analysis. I.M.
performed the carbohydrate analysis. I.C. participated in the discussion of physiological responses.
V.K.M. wrote the paper and A.K.K. helped to prepare the manuscript.

Acknowledgements – V.K.M. and A.K.K. were supported by National Science Centre grant no.
2011/01/B/NZ1/04315. The authors thank Paweł Jedyński and Przemysław Werecki for the excellent
technical assistance, Dr Łukasz Marczak (Institute of Bioorganic Chemistry, Polish Academy of
Sciences in Poznań) for the possibility of performing carbohydrate analysis using GC-MS and the
Laboratory of Microscopy Imaging and Specialized Biological Techniques at University of Lodz,
Poland for the assistance in confocal and transmission electron microscopy. The authors have no
conflicts of interest to declare.

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Supporting Information

Additional Supporting Information may be found in the online version of this article:
Appendix S1. Alternaria brassicicola development on each of the five mature leaves of Brassica
juncea during first 24 h of infection
Appendix S2. Autofluorescence of Brassica juncea leaves during Alternaria brassicicola infection
Appendix S3. Correlations for primary and secondary metabolism parameters in Brassica juncea
leaves during Alternaria brassicicola infection

Figure legends

Fig. 1. Alternaria brassicicola conidia development on the five mature leaves of Brassica juncea
during 20 h of infection. Means ± SE (bars) were obtained from experiment on three plants at each
time point.

Fig. 2. Spreading of necrotic lesions on five mature leaves of Brassica juncea during 96 h of infection
by Alternaria brassicicola. (A) Control leaves; (B) infected leaves after 24 hpi and (C) 96 hpi; (D)
necrosis diameter; p = 0.00049; means ± SE (bars) were obtained from three independent experiments
(n = 3).

Fig. 3. Autofluorescence 3D images of Brassica juncea leaves infected by Alternaria brassicicola on

the border of healthy and infected areas at 48 hpi. (A) 1st (the oldest) leaf; (B) 3rd leaf; (C) 5th (the
youngest) leaf. Images were captured using a confocal microscope. Magenta indicates chlorophyll
fluorescence; green indicates the fluorescence of dying/dead cells or abundance of secondary
metabolites in necrotic cells. Bar = 200 μm.

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 Fig. 4. Autofluorescence of Brassica juncea leaves infected by Alternaria brassicicola at 96 hpi.
Images were captured using a confocal stereo microscope. Magenta indicates chlorophyll
fluorescence; green indicates fluorescence of dying/dead cells or abundance of secondary metabolites
in necrotic cells. Fluorescence intensity was estimated in the middle of chl+cell death images with
Leica LAF-AS software. Lambda scan revealed two peaks of autofluorescence at 620-680 nm
(magenta) and 545-568 nm (green). chl – chlorophyll; bar = 2-2.5 mm.
Accepted Article

Fig. 5. Photosynthetic parameters of Brassica juncea leaves infected by Alternaria brassicicola. (A)
Ratio of chlorophyll a:b, means ± SE (bars) were obtained from four independent experiments (n = 4);
(B) Fv/Fm parameter and (C) non-photochemical quenching (NPQ) parameter, means ± SE (bars) were
obtained from three independent experiments (n = 3). Asterisks indicate a statistically different value
between control and infected leaves at indicated time points according to Duncan`s test (P < 0.05).

Fig. 6. Changes in the chloroplast structure in mesophyll cells of Brassica juncea leaves during
infection with Alternaria brassicicola. (A, A`) control chloroplasts in the 1st and 3rd leaf mesophyll
cells; (B, B`) infected mesophyll cells with hyphae; (C, C`) chloroplasts with large starch grains and
disturbed thylakoids in the 1st and 3rd leaf mesophyll cells at 24 hpi; (D, D`) chloroplasts with huge
starch grains and destroyed outer and inner membranes and thylakoid structure in mesophyll cells of
the 1st and 3rd leaves at 48 hpi; h – hyphae, p – plastoglobule, sg – starch grain; bar (B`, D`) = 10 μm

Fig. 7. Carbohydrates content in Brassica juncea leaves during infection with Alternaria brassicicola.
(A) glucose, (B) fructose and (C) sucrose content; means of six measurements (n = 6) ± SE (bars)
were obtained from two independent experiments. Asterisks indicate a statistically different value
between control and infected leaves according to Duncan`s test (P < 0.05) at indicated time points.

Fig. 8. Temporal changes in secondary metabolites content in each of the five mature leaves of
Brassica juncea during infection by Alternaria brassicicola. (A) Total phenolic compounds content
and (B) flavonoid content, means ± SE (bars) were obtained from three independent experiments (n =
3); (C) glucosinolate content, means of six measurements (n = 6) ± SE were obtained during two
independent experiments. Asterisks indicate a statistically different value between control and infected
leaves according to Duncan`s test (P < 0.05) at indicated time points.

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