Pak. J. Phytopathol., Vol. 34 (02) 2022. 213-220 DOI: 10.33866/phytopathol.034.02.

0799

Official publication of Pakistan Phytopathological Society

Pakistan Journal of Phytopathology

ISSN: 1019-763X (Print), 2305-0284 (Online)
http://www.pakps.com

INDUCED SYSTEMIC RESISTANCE OF OKRA (ABELMOSCHUS ESCULENTUS L.

MOENCH) AGAINST OKRA YELLOW VEIN MOSAIC VIRUS USING AMINO ACIDS AND
ALGAE EXTRACTS
Malik H. Karem, Ali A. A. Haidery
Department of plant protection, college of agriculture, Al-Muthanna University, Iraq.

ABSTRACT

Abelmoschus esculentus L. Moench, more often known as okra, is a popular and widely consumed vegetable that belongs
to the Malvaceae family. It is rich in a variety of essential nutrients, including carbohydrates, proteins, and vitamins. The
okra yellow vein mosaic disease (OYVMD) is one of numerous biotic and abiotic variables that pose a danger to okra
productivity. In order to investigate the impact of amino acid treatments and algal extracts on inducing systemic
resistance in okra against the Okra yellow vein mosaic virus, an experiment was carried out in the fields in the western
areas of Samawa City-Muthanna Governorate, Iraq (OYVMV). Research on the impact of the virus found that amino acid
and algal extract treatments were most effective, leading to the fewest number of infected okra plants. Peroxidase
enzyme, superoxide dismutase, and catalase levels were highest in those who received a combination of amino acids and
algal extract. The duration of 14 days was also longer than the spans of 7 and 21 days. While amino acid and algal extract
therapy for 21 days had the highest total phenolic concentration, it also had the most negative effects.
Keywords: Okra yellow vein mosaic virus, Algal extract, Amino acids, DAS-ELISA.

INTRODUCTION infestations and viral transmission affect the okra

Okra (Abelmoschus esculentus L. Moench) is one of the industry. The family Geminiviridae includes the genus
most important and well-liked vegetables in the world Begomovirus, which includes the Okra Yellow Vein
and is a member of the Malvaceae family (Mubeen et al., Mosaic Virus (OYVMV) (Mubeen et al., 2021). There is a
2021). One of the important vegetable crops grown in sizable viral family called OYVMV that is responsible for
Iraq is okra (Al-Shammary, 2018). The production has widespread crop failures around the world. Caused by a
substantially decreased over the past few years as a virus, symptoms include chlorosis, stunting, chlorosis of
result of a variety of problems, including biotic pressures different degrees, and yellowing of veins and veinlets
from various insects and viral infections (Fekrat and (Venkataravanappa et al., 2012). However, the OYVMV
Shishehbor, 2007; Hussain et al., 2011, 2012, 2014, has a detrimental effect on okra plants, the virus is
2016; Hussain and Mukhtar, 2019; Kassi et al., 2018, widespread throughout the world's okra-growing
2019; Mukhtar and Hussain, 2019; Mukhtar et al., 2013a regions, and the virus infects okra plants at every stage
b, 2014, 2017). The whitefly Bemisia tabaci (Genn.), of development and is to blame for 80–90% of yield
which feeds on okra, is responsible for the propagation losses (Mubeen et al., 2017; Mohanta et al., 2020). Since
of Phyto-viruses such okra yellow vein mosaic virus and there is currently no viricide, viral infections are only
okra leaf curl. Significant crop losses due to whitefly indirectly treated by using insecticides against their
Submitted: August 28, 2022 vectors, the widespread use of insecticides puts the
Revised: October 09, 2022 environment's safety at risk by contaminating the air,
Accepted for Publication: December 12, 2022 water, and soil (Ali et al., 2014). It can be tricky to detect
* Corresponding Author: a virus through the morphological symptoms of the host
Email: malik.hasan@mu.edu.iq plant, as the symptoms of each viral strain differ from
© 2017 Pak. J. Phytopathol. All rights reserved. host to host, and as the symptoms of a plant virus

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Pak. J. Phytopathol., Vol. 34 (02) 2022. 213-220 DOI: 10.33866/phytopathol.034.02.0799

infection can manifest in a wide range of ways chemicals that are used to protect plants. The European
depending on the plant (Lacroix et al., 2016). Thus, Biostimulants Industry Council estimates that
bioassays based on polymerase chain reaction (PCR) and biostimulants are used on more than four million
enzyme-linked immune-sorbent assays (ELISA) have hectares of land in Europe, however other sources place
greatly improved virus detection (Diaz-Lara et al., 2020). the number at more than six million hectares (EBIC
The creation and application of new alternative inputs Biostimulants, 2003; Calvo et al., 2014). Infection
for agricultural production are in high demand. problem was reduced after amino acid spraying was
Researchers have recently been investigating the use of implemented, with reports of a 16% decrease in
organic biostimulants to improve plant growth and Fusarium patch (Microdochium nivale) infection and a
defensive mechanisms (yakhin et al., 2017). Seaweed 20% decrease in Dreschlera leaf spot (Drechslera
extracts, which have been demonstrated to exhibit both siccans) (Radkowski et al., 2020). Biostimulant
phytostimulatory and phytoelicitor qualities, are a treatments had varying effects on all test parameters,
prospective source of biostimulants (Ramkissoon et al., but they were most effective in reducing the imbalance
2017; Rayorath et al., 2008). Although extracts from a caused by PepMV infection, which was practically halved
number of marine algae species have been shown to in the two virus-resistant pepper cultivars (Betti et al.,
have potential as plant biostimulants, Ascophyllum 1992).
nodosum, brown seaweed, is the most significant species MATERIALS AND METHODS
used internationally for commercial extraction (Ali et al., Experiment layout: The effectiveness of inducing
2016; Jayaraj et al., 2008). Inducible defense systems resistance in okra to the okra yellow vein mosaic virus
allow plants to fend off a wide range of pathogens, but was assessed in an experiment (OYVMV). We used the
they are only activated in response to specific signaling Local-Hussainawya type of okra, and we planted the
molecules released by pathogens or by plants subjected seeds in 8–10 cm deep holes in plastic dishes with a
to environmental challenges. Seaweed extracts mixture of one part peat moss to two parts sand.
containing elicitor molecules can boost several inducible Spraying the plants with an algae extract (Quelafert
defensive reactions in plants. Induced resistance occurs quelgreen) and amino acid (Aminoprim) solution at a
in plants after a sequence of events in which the elicitor rate of 0.5 milliliters per liter of water was done after the
attaches to specific receptor sites on the membrane. plants had grown to the size of three complete leaves.
After binding, secondary chemical messengers further The whitefly was then sprayed with Mospilan
amplify a signal, which triggers subsequent defensive (acetamiprid 20%) after being artificially inoculated
mechanisms (Jayaraj et al., 2008). This starts a series of with the virus by pre-feeding on an infected plant. The
chemical processes in the plant that help it become more plants had their soil replaced with plastic pots that were
resistant to pathogen invasion. The plant can defend 22 cm by 24 cm, and they were placed in a tiny area
itself from a wide range of pathogens and pests, (hidden by a boring cloth tent) to prevent pests from
including as bacteria, fungus, parasites, viruses, getting to them. Every day and in accordance with the
nematodes and insects, thanks to this broad-spectrum plants' requirements, the pots are watered, following
resistance (Jayaraj et al., 2008). Induced resistance is the that, tests were taken 7, 14 and 21 days after the virus
outcome of chemical stimulation, which triggers a inoculation. Young symptomatic leaves were gathered
cascade of events including the phenylpropanoid and examined by DAS-ELISA to confirm that the virus
pathway, the assembly of defense signaling molecules, was the source of the symptoms (Clark and Adams,
and the accumulation of antimicrobials including 1977). These were the treatments.
pathogenesis-related (PR) proteins and phytoalexins Table 1. Treatments Applied and details.
(Ramkissoon et al., 2017).
Treatment Detail
As for amino acids, In order to improve growth and
T0 Healthy control
development, they work by enhancing the inherent
T1 Inoculated control
tolerance and resistance of plants to stress and by
T2 OYVMV + Amino acids
stimulating genetic potential (Kumar et al., 2020). As a
result, biostimulants can increase the effectiveness of T3 OYVMV + Algal extract
conventional fertilizers and can replace synthetic T4 OYVMV + Amino acids + Algal extract

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Pak. J. Phytopathol., Vol. 34 (02) 2022. 213-220 DOI: 10.33866/phytopathol.034.02.0799

Screening for OYVMV under natural conditions: The disease was measured using a standardized scoring
plants were grown in an open field where they could be system (Wasala et al., 2019). Because of this, the
exposed to the virus freely, and the severity of the following grades have been given out (Table 2).
Table 2. OYVMV disease symptoms are described for the score.
Symptoms description Severity scale Severity range (%)
Absence of symptoms 0 0
Very mild symptoms 1 1-20
Leaf veins turn yellow, but interveinal areas remain green or normal. 2 20-40
Curly leaves and the entire leaf become yellow. 3 40-60
The entire leaf turns yellow. The margin begins to dry. 4 60-80
Yellowish, malformed pods with completely yellow leaves. 5 80-100
Assays of antioxidant enzymes: Enzyme Extraction: kadhum (2018).
According to the procedure listed below, the POD, SOD Assay for Total phenolic (µg/ml): For the assay of the
and CAT activities in okra leaves were measured. With Total phenolic, the method described by Cl and Indira
the aid of a pestle and mortar, three 0.2 g FW leaf (2016).
samples were collected, using the aid of a pestle and STATISTICAL ANALYSIS
mortar, and ground in liquid nitrogen. After adding and The experiment was designed to be carried out using a
homogenizing 2ml of 50mm ice-cold phosphate buffer randomized complete block design (RCBD). The results
(pH 7.7) and 1mM ethylene diamine teraacetic acid were put through a series of statistical analyses using
(EDTA) was added. Centrifuged for 15 minutes at 4°C at GenStat version 18. (LSD, 0.05).
1500 rpm. The enzyme extract was made from the RESULTS
supernatant. Disease Severity of OYVMV: Disease Severity was
Assay for peroxidase (POD) (U/mg): For the assay of measured and the results were displayed graphically in
the peroxidase enzyme, the method described by Pitotti Figure 1. The inoculation plants (control) comparison
et al. (1994). plants gave the highest infection severity by 41%, while
Assay for superoxide dismutase (SOD) (U/mg): For the treatment (OYVMV + amino acids + algae extract)
the assay of the superoxide dismutase enzyme, the had the lowest disease severity with the virus,
method described by Magnani et al. (2000). amounting to 13%. Disease Severity was at (26% and
Assay for catalase (CAT) (U/mg): For the assay of the 18%, respectively) for the two treatments (OYVMV +
catalase enzyme, the method described by Hadwan and algal extract and OYVMV + amino acids).
50
45 41
40
35
Disease Severity

30 26
25
20 18
15 13
10
5 0
0
Healthy Control Inoculated OYVMV + Amino OYVMV + algal OYVMV + Amino
control acids extract acids + algal
extract
Treatments

Figure 1. Disease Severity of OYVMV in a Glass House Regarding Treatments

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Peroxidase (POD) (U/mg): It also showed the peroxidase concentration of the enzyme after 14 and 21 days (18.05
enzyme (POD) concentration in okra leaves (Figure 2), and 17.32 U/mg). Then followed the (OYVMV + Amino
When compared to the other treatments, the treatment Acids) treatment, which resulted in the lowest
(OYVMV + Amino acids + algal extract) had the greatest concentration of the enzyme after 21 days of all treatments.
20.00
18.00
16.00
14.00
12.00
(U/mg)

10.00
8.00 7day
6.00
14 day
4.00
2.00 21 day
0.00
Healthy Inoculated OYVMV + OYVMV + OYVMV +
Control control Amino acids Ascophyllum Amino acids +
nodosum Ascophyllum
nodosum
Treatment

LSD (0.05) Treat. = 0.023, Times= 0.018 and for Intersection= 0.04
Figure 2. Okra leaf content of peroxidase (POD) (U/mg)
Superoxide dismutase (SOD) (U/mg): During the 14 days, Amino acids + algal extract) (3.58 U/mg), there was no
the highest concentration of the enzyme superoxide significant variation in enzyme concentration between 7 and
dismutase (SOD) was also given to the treatment (OYVMV + 21 days (3.22 and 3.27 U/mg) (Figure 3).
4.00
3.50
3.00
2.50
(U/mg)

2.00
1.50 7day
1.00 14 day
0.50 21 day
0.00
Healthy Inoculated OYVMV + OYVMV + OYVMV +
Control control Amino acids Ascophyllum Amino acids +
nodosum Ascophyllum
nodosum
Treatment

LSD (0.05) Treat. = 0.015, Times= 0.012 and for Intersection= 0.026
Figure 3. Okra leaf content of superoxide dismutase (SOD) (U/mg)
Catalase (CAT) (U/mg): Figure 4 shows that after 14 days, acids treatments in terms of enzyme concentration (3.68
there was no statistically significant difference between the and 3.55 U/mg). However, there was little change in
OYVMV + Amino acids + algal extract and OYVMV + Amino enzyme content between 7 and 21 days in the leaves.

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4.00
3.50
3.00
2.50
(U/mg)

2.00
1.50 7day
1.00
14 day
0.50
0.00 21 day
Healthy Inoculated OYVMV + OYVMV + OYVMV +
Control control Amino acids Ascophyllum Amino acids +
nodosum Ascophyllum
nodosum
Treatment

LSD (0.05) Treat. = 0.148, Times= 0.115 and for Intersection= 0.257
Figure 4. Okra leaf content of catalase (CAT) (U/mg)
Total phenolic (µg/ml): Regarding the concentration of the enzyme concentration for the treatment (OYVMV +
total phenolic in okra leaves (Figure 5), The highest Amino acids) (26.29 µg/ml). There was no significant
concentration after 21 days of treatment was (OYVMV + difference between the concentration of total phenols for
Amino acids + algal extract) (26.97 µg/ml) , followed by the periods of 7 and 14 days for most of the treatments.
30.00
25.00
20.00
(µg/ml)

15.00
10.00 7day

5.00 14 day

0.00 21 day
Healthy Inoculated OYVMV + OYVMV + OYVMV +
Control control Amino acids Ascophyllum Amino acids +
nodosum Ascophyllum
nodosum
Treatment

LSD (0.05) Treat. = 0.72, Times= 0.557 and for Intersection= 1.246
Figure 5. Okra leaf content of Total phenolic (µg/ml)
DISCUSSION (Lacroix et al., 2016).
The severity of symptoms caused by Okra yellow vein Pesticides are harmful to the environment, so scientists
mosaic virus (OYVMV) varies from plant to plant, season are increasingly looking to chemical and natural
to season, whitefly population to whitefly population, materials, such as amino acids (Betti et al., 1992;
and nutritional status of the plant. With no effective Jayaraman et al., 2011) to induce systemic resistance in
viricide against plant viruses, researchers have focused plants. It was determined that a commercially available
on developing resistant plant varieties through selective Ascophyllum nodosum alkaline extract effectively
breeding and the introduction of resistance genes, or on stimulated plant growth and elicited a plant's defense
controlling insect vectors through the use of pesticides mechanism against foliar diseases caused by

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the highest yield was increased by 57% (Ali et al., 2019). 1992. Effects of foliar application of an amino-
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diseases (Jayaraj et al., 2011). biostimulant on the response of pepper seedlings
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ACKNOWLEDGEMENTS EBIC Biostimulants—A sector born from industry
The authors are thankful to the Chairman of the convergence and sustainability-driven innovation.
Department of plant protection for providing necessary In Position on the Revision of Reg (EC)
research facilities. 2003/20031; European Biostimulants Industry
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Contribution of Authors:
Malik H. Karem : Designed the layout and performed experiments and wrote the manuscript.
Ali A. A. Haidery : Editing of manuscript

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