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- Source title: Melanoidins Suppress Cucumber and Tomato Diseases Incited by Pathogens that are Oomycetes, Bacteria and a Virus
- Author(s): Yigal Elad, Dalia Rav-David, Ludmila Tsechansky, Ellen R Graber
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## **ORIGINAL ARTICLE**

# **Melanoidins Suppress Cucumber and Tomato Diseases Incited by** **Pathogens that are Oomycetes, Bacteria and a Virus**


**Yigal Elad** **[1*]** **, Dalia Rav-David** **[1]** **, Ludmila Tsechansky** **[2]** **, Ellen R. Graber** **[2]**


1 Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani
Institute, Rishon LeZion 7534509, Israel
2 Department of Soil Chemistry, Plant Nutrition and Microbiology, Agricultural Research
Organization, Volcani Institute, Rishon LeZion 7534509, Israel

*Correspondence: Yigal Elad elady@volcani.agri.gov.il

Running title: Melanoidins suppress diseases caused by Oomycetes, bacteria and a virus

## **Abstract**

We recently reported that solutions of the nitrogenous compounds melanoidins (M), that are formed
during the final stages of the Maillard Reaction, are inducers of plant resistance (Elad et al. 2026). M
promoted immunity and induced disease resistance through an effect on the transcriptome, primarily
through the salicylic acid pathway. Here we tested the effect of M on plant diseases that are caused
by Oomycetes, bacteria and a tobamovirus. Drench and spray of M suppressed _Pythium_
_aphanidermatum_ damping off in cucumber, cucumber downy mildew ( _Pseudoperonospera_
_cubensis_ ), tomato late blight ( _Phytophthora infestans_ ), bacterial speck ( _Pseudomonas syringae_
pv _. tomato_ ), bacterial canker ( _Clavibacter michiganensis_ subsp. _michiganensis_ ) and the Tobamovirus
Tomato brown rugose fruit virus (ToBRFV) in tomato. In various experiments control was achieved
by concentrations of 0.03 and 0.1% and on older and younger leaves and even in long term
experiments where the M was applied for ca 11 weeks. It can be concluded that the disease resistance
inducer M can suppress soilborne and foliar diseases that are caused by Oomycetes, bacteria and a
virus of the genus Tobamovirus, in addition to the fungal pathogen _Botrytis cinerea_ reported earlier.

Keywords Induced resistance, Foliar disease, Soil borne disease, Bacterial pathogen, Oomycetes,

ToBRFV

## **Introduction**

Melanoidins (M) are nitrogenous compounds that are formed during the final stages of the Maillard
Reaction, originally described in early twentieth century (Maillard 1912). The chemical pathways
involved in the Maillard Reaction were described by J.E. Hodge (1953). The variability in backbone
composition of melanoidins and lack of a distinct molecular structure results in heterogeneity in the
melanoidins that vary depending on the starting reactants and their ratios, ranging from amino acidbased to protein-based (melanoproteins) or polysaccharide-based compounds (Morales et al. 2012).
Recently, we reported that M promoted cellular immunity and disease resistance against the fungal


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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plant pathogen _Botrytis cinerea_ (Elad et al. 2026). M was found to promote immunity and induce
disease resistance through a swift and transient effect on the transcriptome, primarily through the
salicylic acid (SA) pathway, as was demonstrated by transcriptomics and genetic defense mutants
(Elad et al. 2026). In the present research we tested if M affects plant diseases that are caused by
Oomycetes, bacteria and a virus.

The Oomycete _Pythium aphanidermatum_ causes damping off in many plant species (Ben Yephet
and Nelson 1999; Jaiswal et al. 2018) including cucumber (Al � Sa'di et al. 2007; Elad and Chet
1987; Sivan et al. 1984). Induced resistance of _P. aphanidermatum_ by _Pseudomonas chlororaphis_
and _Bacillus subtilis_ was reported in pepper (Nakkeeran et al. 2006). Benzothiadiazole (BTH)
pretreatment of cucumber seeds resulted in reduced damping off in cucumber seedlings (Salari et al.
2025). Pretreatment of cucumber growth medium with biochar resulted in induced resistance to _P._
_aphanidermatum_ in cucumber that was attributed both to chemical and microbiological changes in
the medium (Jaiswal et al. 2018, 2019).

Cucurbits downy mildew is caused by the Oomycete _Pseudoperonospora cubensis_ (Savory et al.
2011). It potentially causes severe epidemics in cucumber (Barnea et al. 2022; Shtienberg et al.
2010). Induced resistance in cucumber against _P. cubensis_ was obtained with nutritional elements
such as P, K, and Mg (Barnea et al. 2022) and silicon (Yu et al. 2010).

Late blight, caused by the Oomycete _Phytophthora solani_, is a major disease in tomato and
potato (Frenkel et al. 2010). In tomato leaves it causes severe epidemics in greenhouse grown crops
and in open fields (Palti and Rotem, 1973; Shtienberg et al. 2010). Control of tomato late blight by
induced resistance was reported for an isolate of _Trichoderma harzianum_ (Chowdappa et al. 2013),
plant growth-promoting rhizobacteria (PGPR, Yan et al. 2002) and [beta]-Aminobutyric acid
(BABA, Cohen et al. 1994).


Tomato bacterial speck, caused by the bacterial pathogen _Pseudomonas syringae_ pv. _tomato_, is a
gram-negative bacterium that epidemiologically is specific to this crop under field conditions (Falik
et al. 1983; Preston, 2000). Reports on induced resistance against tomato bacterial speck are limited.
Yoo et al. (2018) reported that an isolate of _Aspergillus terreus_ that is a plant growth promoter which
controls bacterial speck disease by inducing resistance in tomato plants. The endophytic
bacteria _Bacillus pumilus_ and _B. amyloliquefaciens_ induce resistance against bacterial speck in
tomato plants. Induced resistance in this case had a slight negative effect on plant growth parameters
such as plant height and plant dry weight (Lanna-Filho et al. 2017).

_Clavibacter michiganensis_ subsp. _michiganensis_, the causal agent of bacterial canker and wilt of
tomato, is an important bacterial pathogen worldwide that also caused severe epidemics in Israel
(Blank et al. 2016). It is a gram-positive bacterium that causes wilting, leaf necrosis, and
characteristic small spots (Kleitman et al. 2008; Savidor et al. 2012). Among the inducers of
resistance that were reported to reduce bacterial canker of tomato are acibenzolar-S-methyl (ASM)
(Soylu et al. 2003), an isolate of a rhizobacterium _Pseudomonas sp._ (Takishita et al. 2018) and the
plant growth promoting bacteria _Bacillus subtilis_, _Pseudomonas fluorescens_ or _Azotobacter_
_chroococcum_ (Kolomiiets et al. 2019).

Tomato brown rugose fruit virus (ToBRFV) is a virus of the _Tobamovirus_ genus that was first
described in 2014 in Israel (Luria et al. 2017) and Jordan in 2015 (Salem et al. 2016). The main hosts
of ToBRFV are tomato and peppers (Salem et al. 2023). The virus causes symptoms including
mosaic and distortion of leaves and brown, wrinkly spots (rugose) on fruits and it spreads short
distances by mechanical contact (Luria et al. 2017; Salem et al. 2016, 2023). _Pseudomonas_
_fluorescens_ and _Burkholderia gladioli_ isolates sprayed on tomato plants enhanced defense enzyme
activities, upregulated gene expression, and reduced symptom severity while their combination was


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superior to the treatments with the individual bacteria suspensions (Zohoursoleimani et al.
2025). The α -aminophosphonate ester Dufulin, an activator of the salicylic acid (SA) pathway,
controlled ToBRFV in tomato plants (Wang et al. 2026).

In view of the ability of M to induce resistance against a fungal pathogen (Elad et al. 2026), we
tested if the resistance inducer may also affect the above-mentioned pathogens on cucumber and
tomato. The tests of M solutions were aimed at the suppression of the soilborne _P. aphanidermatum_
and foliar _P. cubensis_ Oomycetes pathogens of cucumber, and in tomato, the Oomycete _P. solani_, the
bacterial pathogens _P. syringae_ pv. _tomato_ and _C. michiganensis_ subsp. _michiganensis_ and the
_Tobamovirus_ ToBRFV.

## **Materials and methods**

**Plants' growth and maintenance**

Plants were grown in either 1 L or 10 L pots containing a growth mixture of 7:3, v/v peat–tuff (sorted
to ≤ 8 mm) referred-to below as 'plants-growth medium'. Fertigation was applied 2 to 3 times daily
via drip irrigation using 5:3:8 NPK fertilizer (120:30:150 mg L � ¹ N:P:K; EC 2.2 dS m � ¹),
maintaining 25–50% drainage (Elad et al. 2010, 2012, 2026; Gupta et al. 2024; Mehari et al. 2015).
The experiments with plants were held in a 20-mesh net house (April–October 2018–2024) or in a
glasshouse with 25% shade netting and whitewash (Livnat, Tapazol, Israel) during the rest of the
year. Day/night temperatures ranged from 18–33°C. All net house and glasshouse experiments were
carried out at the Volcani Institute, Rishon LeZion, Israel (31°58 ′ 09 ″ N, 34°48 ′ 02 ″ E). The plants
were maintained pest- and pathogen-free until treatments were applied (Elad et al. 2010, 2012, 2026;
Gupta et al. 2024; Mehari et al. 2015).

Tomato

Tomato ( _Solanum lycopersicum_ ) cv. Ikram (Syngenta, Israel) seedlings were obtained from Hishtil
Nurseries (Israel) at age of 4 to 5 weeks post sowing. The plants were transplanted to pots and grown
for 4–6 additional weeks before being exposed to pathogens. The tomato plants that were used in
short term experiments were grown in 1 L pots and the plants used in commercial-like long-term
experiments were grown in 10 L containers (Elad et al. 2026; Gupta et al. 2022a,b, 2024). In both
cases there was one tomato plant in each pot, and it was held vertically by clipping it with plastic
rings to bamboo sticks.

Cucumber

Cucumber ( _Cucumis sativus_ ) cv. Manny (Genesis Seeds, Israel) was grown in 1 L pots. Cucumber
plants were used as seedlings for _Pythium aphanidermatum_ assays. The cucumber seeds were seeded
in pots containing pathogen free growth medium (5 seeds per pot), and one day after emergence the
plants were transplanted into infested medium as described below (Jaiswal et al. 2018; Philosoph et
al. 2018).

**Plant diseases inoculation and evaluation**


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Cucumber damping-off was incited by using millet seeds inoculum of _Pythium aphanidermatum_ .
For this purpose, _P. aphanidermatum_ was cultured on PDA and grown at 25°C. Mycelium discs
were transferred to autoclaved millet in 250 ml Erlenmeyer flasks, and the mixture was incubated
for 5 days. Once the mycelium occupied the seeds it was homogenized and mixed into the plantsgrowth medium at a concentration of 0.25% (w/w). Seedlings were transplanted into the infested
plants-growth medium, and mortality was recorded every 2 days. Typical death of seedlings was
observed and documented as damping off mortality (Jaiswal et al. 2018; Philosoph et al. 2018).

_P. apanidermatum_ linear growth was recorded in PDA Petri plates. Mycelium discs from 3
days old cultures was inoculated on PDA plates amended with M in concentrations of 0.01, 0.03,
and 0.1%. The plates were incubated at 25°C and the diameter of colonies was recorded one and
two days later. The area of the colony of the oomycete was calculated.

Cucurbits downy mildew ( _Pseudoperonospora cubensis_ ) developed naturally on 1 to two
months old cucumber plants during fall, winter and spring (November to April) under net-house
conditions and was evaluated for severity as leaf coverage (0–100%) with typical downy mildew
symptoms (Barnea et al. 2022).

Tomato late blight ( _Phytophthora solani_ ) developed naturally during fall, winter and spring
(November to April) under net-house and greenhouse conditions and was evaluated for severity as
coverage (0–100%) with typical late blight symptoms (Frenkel et al. 2010; Shtienberg et al. 2010).

Tomato bacterial speck ( _Pseudomonas syringae_ pv. _tomato_ ) developed naturally during fall,
winter and spring (November to April) under net-house and greenhouse conditions and was
evaluated for severity as coverage (0–100%) with typical bacterial speck symptoms (Bashan et al.
2002; Falik et al. 1983).

Bacterial canker ( _Clavibacter michiganensis_ subsp. _michiganensis_ ) was cultured on LB agar at
28°C, suspended in water (5 × 10 � CFU/mL), and sprayed onto tomato plants (Savidor et al. 2012;
Verma et al. 2026). Plants were maintained at 25 ± 2°C, and disease severity was rated on a 0–

100% scale.

Tomato brown rugose fruit virus (ToBRFV) was inoculated artificially. Israeli isolate of
ToBRFV, ToBRFV-IL (KX619418.1) was used for ToBRFV infection. Tomato plants were sprayed
with carborundum powder suspension prior to inoculation, the plants were let to passively dry and
then the leaves were rubbed with a phosphate buffer solution (0.01 M, pH 7.0) supplemented with
crushed leaves from the ToBRFV-infected tomato plants. The severity of ToBRFV symptoms were
evaluated on a scale of 0-100% (Alon et al. 2021; Klap et al. 2020). RT-PCR analysis was carried
out according to Alon et al. (2021).

**M synthesis and application**

The melanoidins were synthesized by means of Maillard reaction in a dry synthesis method at 150°C
using L-glutamic acid and D-glucose. Powders of the ingredients were mixed with each other at an
equimolar ratio and ground together as described recently (Elad et al. 2026). The M powder was
ground, placed in hermetically sealed vials, and kept in a desiccator with silica gel at room
temperature until used (Elad et al. 2026). Plants were sprayed or drenched with M water solutions at
concentrations of 0.03 and 0.1%. Spray applications were performed using a 1 L hand sprayer
calibrated to emission of fine droplets until full canopy coverage was achieved. The solution was
applied at volume of 8–15 mL per each mature plant depending on plant size. Drench applications


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consisted of 10–30 mL per pot, covering the volume of root zone with ~5% drainage (Elad et al.
2026).

The control of _P. aphanidermatum_ damping off by M was compared with a common fungicide.
The medium was drenched once with a fungicide, 0.25% Dynon in water drench, containing 722 g/L
Propamocarb HCl, Bayer, Germany. The inducer of resistance monopotassium phosphate (MKP;
KH � PO �, ICL Haifa, Israel) was used in one experimental set (Reuveni et al. 1993; Elad et al.
2021).

**Calculations and statistics**

Percentage data were arcsine-transformed prior to analysis. Area under the disease progress curve
(AUDPC) or area under mortality progress curve (AUMPC) were calculated where applicable.
Standard errors (SE) are presented as bars in the figures. Replication ranged from 5 to 6 per
treatment. Data (disease severity, AUDPC, and AUMPC) were analyzed by ANOVA followed by
Tukey–Kramer HSD test, using JMP software (SAS Institute, Cary, NC, USA; α = 0.05). Different
letters indicate significant differences among treatments.

Disease reduction (DR, %) was calculated as:
DR = 100 – 100 × (Tn / Tcon), where Tn is the disease level in treated plants, and Tcon is the
untreated control. DR was calculated for each replicate and averaged for each treatment.

# **Results**

**Effect of M on a soilborne Oomycete –** _**Pythium aphanidermatum**_ **damping off in cucumber**
**seedlings**

_P. aphanidermatum_ damping off in the untreated control reached 41.25% incidence (Fig. S1). Spray
of M at concentrations of 0.03 and 0.1% significantly reduced _P. aphanidermatum_ damping off
during days 10 and 17; at two of the dates the 0.1% M was significantly better than 0.03% M in
reducing damping off (Fig. 1a). The results in progress curves presented in Fig. S1 were calculated in
order to analyze the area under mortality progress curve (AUMPC). The AUMPC was significantly
reduced by 52.2 and 62.7% by 0.03 and 0.1%, respectively (Fig. 1A). _P. aphanidermatum_ damping
off reached 45% in the untreated control 18 days after the initiation of the M treatments that are
summarized ni Fig. 1b. M drench and spray reduced _P. aphanidermatum_ damping off in young
cucumber plants by 67.7 and 76.0%, respectively, and dynone (propamocarb) reduced it by 97.0%
(Fig. 1b).


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**Fig. 1** Effect of M on Oomycetes incited soilborne disease in cucumber – Pythium damping off. **a** . M
spray was applied at concentrations of 0.03 and 0.1% on the day of seedlings transplanting in the
_Pythium aphanidermatum_ amended growth mixture and 7 days afterwards as described in Fig. S1
and the area under the mortality progress curve (AUMPC) of data presented in Fig. S1 was
calculated through 17 days after transplanting. **b** . M was applied by spraying and by drenching and
the chemical pesticide, dynone (Propamocarb HCl, Bayer) was drenched in cucumber seedlings to
control the mortality of cucumber seedlings due to _P. aphanidermatum_ in a contaminated growing
medium. M was applied at 0, 7, and 14 days after seeding and dynone was applied at 0 and 7 days.
(AUMPC) through 18 days after seeding. Damping off was evaluated every 2 to 3 days and
presented as percent of all plants in each pot. Percent data at each evaluation date (Fig. S1) and
AUMPC values in each graph followed by a different letter are significantly different according to
one-way ANOVA with Tukey’s HSD test. Default significance levels were set at α = 0.05. Bars =
SEs.

**Effect of M on the growth of** _**Pythium aphanidermatum**_ **in culture**

The M was mixed in PDA growth medium in concentrations of 0.01, 0.03, and 0.1%, and the
pathogen's mycelium growth on it was followed for 2 to 3 days. After two days _P. aphanidermatum_
reached 41.8 cm [2] in the non-amended medium and 37.4 to 44.2 cm [2] in the amended media with no

significant difference between treatments _._

**Effect of M on a foliar Oomycetes** **pathogen –** _**Pseudoperonospera cubensis**_ **downy mildew**
**(PcCDM) in cucumber**

In a short-term experiment, we tested if 0.1% M applied by spray and drench affects _P. cubensis_
downy mildew (PcCDM) on cucumber leaves (Fig. S2). M treatments significantly reduced the
severity of PcCDM by 70.8 to 73.8%, respectively (Fig. S2). M spray and drench treatments of 0.03
and 0.1% were applied under commercial-like conditions on a weekly basis. PcCDM was
significantly reduced by 41.1-61.3, 49.1-61.4 by the four treatments at 41, 58, and 71 days after
treatments' initiation, respectively, while at 71 days only the 0.1% M drench and spray treatments
significantly reduced the disease severity, achieving 51.8-53.1% reduction (Fig. 2a). In a second
commercial-like setup, M 0.1% spray and drench treatments were compared with the Mono
potassium phosphate (MKP) disease resistance inducer. PcCDM evaluated at 61 days after


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treatments initiation was 41.2 and 16.8% on leaves 8 and 13, respectively (Fig. 2B). The disease
severity was reduced by 60.6 and 76.1, 93.9, and 100 and 91.5 and 100% the MKP, and M spray and
drench treatments on the respective leaves, respectively (Fig. 2b). The extent of efficacy on the two
leaves was similar.





























**Fig. 2** Effect of M on an Oomycetes incited foliar disease in cucumber – Control of
_Pseudoperonospora cubensis_ cucumber downy mildew (PcCDM). **a** . Control of naturally developed
PcCDM by 0.03 and 0.1% M spray and drench in a commercial-like set up where applications were
carried out on a weekly basis and disease severity was evaluated at 41, 58, and 71 days after
treatments initiation. **b** . Control of naturally developed PcCDM by 0.1% M drench and spray and by
MKP (KH 2 PO 4, ICL Haifa, Israel) spray, applied on a weekly basis. PcCDM was evaluated on two
leaves at 61 days after sprays initiation. Severity of PcCDM was evaluated on a 0 to 100% scale, in
which 0 = healthy leaves and 100% = leaves completely covered by the damage symptoms. Values
in each date (A) and each leaf (B) followed by a different letter are significantly different according
to one-way ANOVA with Tukey’s HSD test. Default significance levels were set at α = 0.05. Bars =
SEs.

**Tomato late blight (** _**Phytophthora infestans**_ **) affected by M**

Late blight ( _P. infestans_ ) occurred naturally on tomato plants and reached in the untreated control,
19.4% severity 13 days after the initial M treatment. The M 0.1% spray that was applied twice (at
days 0 and 5) significantly reduced disease severity on stem and leaves at all evaluation dates (Fig.
S3); It significantly reduced the severity as calculated for AUDPC through days 1 to 13 by 51.4%
(Fig. 3a). One application of 0.03% M spray reduced the tomato late blight by 57.3% (Fig. 1b). Late
blight on tomato plants in an experiment that was carried out under commercial-like conditions was
treated weekly with 0.03 and 0.1% M drenches and sprays. The disease was significantly reduced
through 76 days after initiation at the 0.1% spray treatment by 46.1 to 76.0%; at the 0.1% drench by


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63.6 to 100%; at the 0.03% spray by 75.8 to 94.7%. The 0.03% drench significantly reduced the
disease by 57.4 to 86.7% during days 33 to 62 after treatments initiation (Fig. 3c). The calculated
AUDPC was significantly reduced by 51.9, 74.3, 85.5, and 58.3% by M 0.1% spray and drench and
by 0.03% M spray and drench, respectively (Fig. 3d).























































**Fig. 3** Effect of M on late blight caused by _Phytophthora infestans_ in tomato plants. **a** . Plants were
treated by 0.1% M spray at days 0 and 5 and disease severity was evaluated until day 13 after the
initiation of M applications as described in Fig. S3 and area under disease progress curve (AUDPC)
was calculated. **b** . Plants were treated by 0.03% M spray once and disease severity was evaluated at
day 5 after the initiation of M applications. **c** . Tomato plants were treated 11 times on a weekly basis
by M spray and drench at concentrations of 0.03 and 0.1% and disease severity was evaluated 4
times until day 76 after treatments initiated. **d** . AUDPC was calculated for the data on treatments
described in **c** . Severity of late blight was evaluated on a 0 to 100% scale, in which 0 = healthy
leaves and 100% = leaves and stems completely covered by the damage symptoms and AUDPC was
calculated as mentioned above. Values in each date and each graph followed by a different letter are
significantly different according to one-way ANOVA with Tukey’s HSD test. Default significance
levels were set at α = 0.05. Bars = SEs.

**M controls bacterial speck (** _**Pseudomonas syringae**_ **pv** _**. tomato**_ **) on tomato canopy**

Bacterial speck ( _P. syringae_ pv _. tomato_, PstBS) occurred naturally on the tomato plants. The disease
was suppressed with 0.1% M drench and spray applications at 11 and 13 days in a similar manner on
lower and upper leaves (Fig. 4a). Disease severity in the untreated control reached 52.5% in an


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experimental set in which the M was treated with 0.03% concentration (Fig. S4). In this experiment
the severity of disease was significantly reduced at dates 6 through 13 by the 0.03% M drench and
spray treatments (Fig. S4). Disease severity, as calculated for AUDPC, was reduced by 77.5-80.0%
and 51.1-61.9 by the M concentrations 0.1% (Fig. 2b) and 0.03% (Fig. 2c), respectively.

In another experimental setup PstBS severity was first evaluated 10 days after the first
application of M (spraying of 0.1% M) and it was evaluated until 11 days later, scoring 37.3%
severity on the whole plants in the untreated control. The M spray treatment reduced the disease by
49.4, 40.5, 72.0, and 51.1% on the lower, middle, and upper leaves and the whole tomato plants,
respectively (Fig. 2D), revealing a better disease reduction on the upper leaves where disease in the
untreated control was lower than on the middle and upper leaves (21.4, 32.6, and 57.9%, respectively
in upper, middle and lower leaves).































































**Fig. 4** Effect of M on the severity of _Pseudomonas syringae_ pv _. tomato_ bacterial speck (PstBS)
naturally developed on leaves of tomato plants. **a** - **c** . Effect of M spray and drench applied at 0 and 4
days after experiment initiation at concentrations of 0.1% ( **a,b** ) and 0.03% ( **c** ) and evaluated through
13 days. Disease was evaluated on lower and upper leaves at two dates ( **a** ) and are also presented as
area under disease progress curve (AUDPC) for whole plants treated by 0.1% M and evaluated for
PstBS through 13 d ( **b**, details described in Fig. 4 **a** ), and AUDPC of disease severity through 13 days
in untreated plants and plants treated by M 0.03% drench and spray ( **c**, detailed results are in Fig.
S4). **d** . Effect of 0.1% M applied by spraying at 0, 7 and 14 days on the severity of PstBS on lower,
middle, and upper leaves and on whole plants (averaged data of the 3 leaf levels=whole plant) as
evaluated through 21 days and calculated for AUDPC. Severity of PstBS was evaluated on a 0 to
100% scale, in which 0 = healthy leaves and 100% = leaves completely covered by the damage
symptoms. AUDPC was calculated through 13 days ( **b** and **c** ) and through 21 days ( **d** ). Values in


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each date and leaf height ( **a** ), graph ( **b**, **c** ) and in each leaf level ( **d** ) followed by a different letter are
significantly different according to one-way ANOVA with Tukey’s HSD test. Default significance
levels were set at α = 0.05. Bars = SEs.

**Effect of M on** _**Clavibacter michiganensis**_ **subsp.** _**michiganensis**_ **bacterial canker (CmmBC) in**

**tomato**

The bacterial canker disease developed after artificial inoculation with suspension of _C._
_michiganensis_ subsp. _michiganensis_ . Typical symptoms that developed on the leaves consisted of
small necrotic dots that spread over the leaflets area (Fig. 5a) and dry rot that spread on the leaflets'
edges (Fig. 5b). The small dots' symptoms were not affected by the M treatments at the early
evaluation of 8 days, and it was significantly reduced on day 11 by 48.9 and 38.5% by the 0.1% M
drench and spray, respectfully (Fig. 5a). The edges dry rot evaluated on day 8 was only 37.5%
reduced by the M spray while on day 11 both treatments significantly reduced their severity by 63.0
and 60.1%, respectfully (Fig. 5b).





























**Fig. 5** Effect of M 0.1% drench and spray applied at days 0 and 3 on the severity of _Clavibacter_
_michiganensis_ subsp. _michiganensis_ bacterial canker (CmmBC) on tomato leaves. Bacterial
suspension was used at day 0 to artificially infect the plants. Symptoms consisted of dots that were
spread on the leaf blade ( **a** ) and spreading rot that developed on leaflet edges ( **b** ). Severity of each of
the typical symptoms was evaluated at two dates (days 8 and 11) on a 0 to 100% scale, in which 0 =
healthy leaves and 100% = leaflets completely covered by the damage symptoms. Values in each
date and symptom type followed by a different letter are significantly different according to one-way
ANOVA with Tukey’s HSD test. Default significance levels were set at α = 0.05. Bars = SEs.

**M affects Tomato brown rugose fruit virus (ToBRFV)**
Tomato brown rugose fruit (ToBRFV), Tobamovirus, is transmitted through mechanical contact and
that was the way of its introduction to the tomato plants in our experiments. Treatments of 0.1%
spray and drench were carried out 3 days before infection, on the infection day, and 8 days later. The


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severity of the ToBRFV symptoms in tomato plants of the untreated control reached 36.0%, 25 days
after infection (Fig. 6 **a** ). Disease symptoms were reduced significantly and similarly until day 21
after treatments initiation while at day 25 the severity reduction was 63.9 and 33.3% in the drench
and spray treatments, respectively, and the drench treatment was significantly superior to the spray
treatment (Fig. 6 **a** ). The AUDPC calculation revealed 37.2 and 62.5% reduction, respectively, in the
spray and drench treatments (Fig. 3 **b** ).

The level of ToBRFV presence in samples of leaves taken in the experiment treatments was 60,
40, and 20% in the untreated control, M spray and M drench, respectively, revealing an effect of
inhibition of virus presence and multiplication in the leaves by M treatment.

































**Fig. 6** Effect of M on the tomato viral disease Tomato brown rugose fruit Tobamovirus (ToBRFV).
M (0.1%) spray and drench treatments were carried out 3 days before infection, on the infection day,
and 8 days later. Infection was carried artificially and the ToBRFV symptoms on the leaves were
evaluated through 25 days after infection. The severity of symptoms on a 0 to 100% scale, in which 0
= healthy leaves and 100% = leaves completely covered by the viral symptoms. AUDPC was
calculated through days 10 to 25. Values in each date ( **a** ) and graph ( **b** ) followed by a different letter
are significantly different according to one-way ANOVA with Tukey’s HSD test. Default
significance levels were set at α = 0.05. Bars = SEs.

## **Discussion**

We recently reported that M solutions affected the severity of the necrotrophic fungal plant pathogen
_Botrytis cinerea_, the causal of gray mold in tomato (Elad et al. 2026). Here we demonstrate that M
also suppresses diseases that are caused by pathogens that do not belong to the fungal kingdom. The
pathogens that were controlled are the Oomycetes _Py. aphanidermatum_ that causes damping off in
cucumber (Figs 1 and S1), foliar diseases caused by Oomycetes downy mildew in cucumber ( _Ps._


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_Cubensis_, Figs 2 and S2) and late blight in tomato ( _Ph. infestans_, Figs 3 and S3). In tomato M also
controlled the bacterial diseases bacterial speck caused by _Ps. syringae_ pv _. tomato_ (Figs 4 and S4)
and bacterial canker caused by _C. michiganensis_ subsp. _michiganensis_ (Fig. 5). Interestingly, the
viral disease ToBRFV was also affected by M in tomato plants (Fig. 6).

M is not toxic to the pathogens as was shown here for _Py. aphanidermatum_ in a culture medium
and shown earlier for _B. cinerea_ (Elad et al., 2026). We reported that M activity is due to promotion
of cellular immunity and by induction of disease resistance against _B. cinerea_ (Elad et al. 2026). M
promotes immunity and induces disease resistance through a swift and transient effect on the
transcriptome, primarily through the salicylic acid (SA) pathway, as was demonstrated by
transcriptomics and genetic defense mutants (Elad et al. 2026).

Induced resistance is commonly divided into induced systemic resistance (ISR) and systemic
acquired resistance (SAR) (Walters and Heil 2007). SAR biosynthesis pathway generally relies on
SA, which has been shown to increase in the plant tissues, during pathogenesis and involves the
activation of pathogenesis-related (PR) genes (Yalpani et al. 1991). SA (as well as jasmonic acid that
is related to ISR) (Gupta et al. 2023; Pieterse et al. 1998; Thulke and Conrath, 1998), SA analogs
such as benzothiadiazole (BTH) (Leibman-Markus et al. 2023; Meller Harel et al. 2014; Perazzolli et
al. 2008), and several amino acids including phenylalanine, glutamine (Gupta et al. 2020; Liao et al.
2024; Oliva et al. 2020), are among the reported inducers of plant immunity. Systemic activity of M
was demonstrated here in suppression cases of _Py. aphanidermatium_ damping off (Fig. 1B), _Ps._
_cubensis_ cucurbits downy mildew (Figs 2 and S2), _Ph. infestans_ late blight (Fig. 3 c,d), _Ps. syringae_
pv _. tomato_ bacterial speck (Figs 4a-c and S4), _C. michiganensis_ subsp. _michiganensis_ bacterial
canker (Fig. 5) and ToBRFV (Fig. 6).

M induced resistance not only against foliar diseases but also against a soilborne disease that
causes damping off in cucumber (Figs 1 and S1) as was also shown in pepper seedlings with two
biocontrol bacteria that induce resistance (Nakkeeran et al. 2006), BTH pretreatment of cucumber
seeds (Salari et al. 2025) and pretreatment with biochar (Jaiswal et al. 2018, 2019). BTH is a SAR
inducer that activates the SAR signal transduction pathway at the site of or downstream of SA
accumulation (Friedrich et al. 1996) while also M induces resistance through the SA pathway (Elad
et al. 2026).

In the case of _Py. aphanidermatum_ damping off, we compared M with a common chemical,
dynone (propamocarb) that statistically was like the spray treatment, but was significantly better than
the drench treatment (Fig. 1). MKP is an inducer of resistance that was tested in cucumber crops
(Reuveni et al. 1993; Elad et al. 2021). In the present study, cucumber downy mildew was
suppressed better by M than by MKP on leaves of two ages at 61 days after initiation of the
applications (Fig. 2b).

Comparison of drench versus spray application methods and two concentrations, 0.03 vs. 0.1%,
revealed that both application methds and concentrations were similarly effective in most cases. The
exceptions were late blight, where the results were inconsistent (Fig. 3d), and the last evaluation date
(day 25) of ToBRFV, where the spray application was significantly less effective than the drench
(Fig. 6a). Yet, overall, there was no significant difference in the calculated AUDPC between drench
and spray effect for ToBRFV (Fig. 6b).

ToBRFV is a Tobamovirus that is transmitted by mechanical contact (Luria et al. 2017; Salem et
al. 2016, 2023). There are no reported control agents for it, and few effective resistance inducers.
Resistance inducers for ToBRFV include sprayed isolates of _Ps._ _fluorescens_   and _Burkholderia gladioli_ on tomato plants (Zohoursoleimani et al. 2025) and the activator of the SA


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pathway, α -aminophosphonate ester Dufulin (Wang et al. 2026). Here we showed that M also has
this activity (Fig. 6).

M significantly reduced bacterial speck better on younger leaves than on medium age and older
leaves (Fig. 4d). Better activity in the younger leaves may be because of longer exposure of their
tissue to the signals of induced resistance than the exposure of the leaf tissue to the pathogen
infection and disease development while in older leaves the exposure to induced resistance signals is
similar to the duration of exposure to the pathogen. Interestingly, the induction of resistance by M
occurred also in experiments that were carried out under commercial-like conditions and lasted even
for 9 to 11 weeks of repeated applications as was demonstrated for downy mildew in cucumber (Fig.
2 A) and late blight in tomato (Fig. 3c,d).
Inducers of resistance in various plant species – pathogen types are chemicals such as nutritional
elements (Gupta et al. 2022b), BABA (Cohen et al. 1994), Dufulin (Wang et al. 2026) and ASM
(Meller Harel, 2014; Perazzoli et al. 2008), fungi and bacteria such as biocontrol control agents
(Gupta et al. 2022b, 2024; Meller Harel, 2014; Perazzoli et al. 2008) and PGPRs (Yan et al. 2002),
and physical treatments like root zone warming (Gupta et al. 2021). We report here the induction of
resistance by a chemical inducer, M, against the various Oomycetes, bacterial and viral pathogens in
cucumber and tomato plants. Reports in the literature regarding the type of inducers of the diseases
that are reported here as targets of M vary with the specific pathogen, as follows. Similarly to M as
chemical inducer, induction of resistance towards _Py. aphanidermatum_ damping off was achieved
with inducers of chemical nature such as biochar (Jaiswal et al. 2018, 2019) and BTH (Salari et al.
2025); _Ps. cubensis_ downy mildew was suppressed with chemical inducer such as nutritional
elements (Barnea et al. 2022) and silicon (Yu et al. 2010); Induction of resistance against _Py. solani_
late blight was achieved with the chemical BABA (Cohen et al. 1994); We did not identify chemical
inducing resistance for Ps. _syringae_ pv. _tomato_ bacterial speck but the chemical inducer ASM was
reported for _C. michiganensis_ subsp. _michiganensis_ bacterial canker; and the chemical Dufulin
induced resistance against ToBRFV in tomato plants (Wang et al. 2026). All these cases of
chemicals, including M, involve induced resistance that primarily is active through the SA pathway
(Cohen et al. 1994; Elad et al. 2026; Gupta et al. 2022b; Meller Harel, 2014; Wang et al. 2026).

In conclusion, M treatments to tomato and cucumber plants resulted in suppression of diseases
caused by Oomycetes, bacteria and a virus. M was found to act as a robust systemic inducer of SAR
that leverages the host’s innate priming mechanisms to provide a broad-spectrum resistance. M is
easily produced in a simple synthesis method, and it has high water solubility, so solutions of this
resistance inducer are ideal for environmentally friendly disease management. M application by
spray to the plant's canopy and by drenching the roots were both effective in controlling foliar and
soil-borne pathogens. M operates not through direct antimicrobial toxicity through the systemic
activation of the host's internal defense machinery (Elad et al. 2026).

## **Authors Contributions**


**Yigal Elad** : Writing – review & editing, Writing – original draft, Supervision, Methodology,
Investigation, Formal analysis, Conceptualization. **Ellen R. Graber** : Writing – review & editing,
Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.


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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
[available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/)


**Dalia Rav-David** : Methodology, Investigation, Formal analysis. **Ludmila Tsechansky** :
Methodology, Investigation.

## Acknowledgments We thank the Israel Innovation Authority and Adama Agricultural Solutions


Ltd. Israel, for partial support of this work, and members of the Elad and Graber research groups for
continuous support. We thank Dr. Omer Frenkel, Dept of Plant Pathology – Volcani Institute for
support, advice and supply of _Py. aphanidermatum_ inoculum, Dr. Daron Teper Dept of Plant
Pathology – Volcani Institute for supplying _C. michiganensis_ subsp. _michiganensis_ suspension, Prof.
Maya Bar Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva for long
term assistance and advice, and Mr. Menachem Borenshtein, and Mr. Eyal Glantz, Dept of Plant
Pathology for the technical support in the greenhouses.

## Funding This work was partially supported by the Israel Innovation Authority (Nitzan grant No.

20-10-0041) and Adama Agricultural Solutions Ltd. Israel.

## Declaration of competing interest We are not aware of a conflict of interest. The authors

declare the following financial interests/personal relationships which may be considered as potential
competing interests: Ellen Ruth Graber, Dalia Rav-David, and Yigal Elad are inventors in a series of
patents belonging to the families of PCT patent application No.s WO2020261273, entitled
"Melanoidin compositions for protecting crops from nonfungal pests", and WO2014111932, entitled
"Melanoidins and their use for improving properties of plants", which include data described in this

paper.

## Data Availability Statement The authors declare that the data supporting the findings of this

study are available within the paper and its Supporting Information files. Raw data is available from
the corresponding author upon reasonable request.
## Research involving human participants and/or animals This article does not contain

any studies with human participants or animals performed by any of the authors.

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