Journal of Phytopathology and Pest Management 9(1): 32-40, 2022
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Hoda A. M. Ahmed,
E-mail: hudafatah@yahoo.com
32
Copyright © 2022
Management of Phytoplasma associated with sesame
(Sesamum indicum L.) in Assiut governorate, Egypt
Hoda A. M. Ahmed1*, Amal I. Eraky2
1Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
2Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt
Abstract
Keywords: sesame, symptoms, Phytoplasma detection, varieties, irradiation.
Phytoplasma associated with sesame can cause serious economic losses in sesame
production in Assiut governorate, Upper Egypt. Various primary symptoms
indicating phyllody disease were noted, including proliferation (witches’ broom),
yellowing, and the transformation of capsules into flowers, resulting in a significant
decrease in sesame yield. Phyllody symptomatology and incidence were studied in
three sesame varieties (Giza 32, Shandawel 3, and Sohag 1). Four irradiation
treatments It was irradiated using Cobalt 60 at different doses (150, 200, 250, and
300 Gy of gamma rays). Data showed that Shandawel 3 was categorized as a
moderately resistant cultivar, while Sohag 1 was grouped as resistant and Gize 32
was categorized as moderately susceptible. This study also revealed that all
irradiation treatments (150, 200, 250, and 300 Gy) reduced the percentage of
infected rate and disease severity caused by phytoplasma. The study recommends
that using resistant varieties is an efficient and sustainable approach to controlling
susceptibility to phytoplasmas in sesame.
Ahmed Hoda & Eraky Amal, 2022
33
1. Introduction
Sesame (Sesamum indicum L.) of the
Pedaliaceae family serves as an ancient oilseed
crop. Sesame is considered to have both
nutritional and medicinal values. Sesame
plants suffer from several fungal, viral,
bacterial, and phytoplasma diseases.
Phytoplasmas are cell-wall-less bacteria that
have very small genome sizes and are
considered among the smallest self-replicating
living organisms (Bertaccini et al., 2014).
Phytoplasma can induce diverse types of
symptoms in sesame plants. The unique
symptoms induced by phytoplasma infection
are stunting, altering the color of leaves from
green to yellow, and the transformation of the
floral parts into leafy structures containing no
capsules or seeds. The most prevalent
symptoms of sesame are phyllody and witch's
broom. In phyllody symptoms, the flowers of
the infected plants turn similar to leaves, while
in witches 's broom symptoms, the leaves of the
infected sesame plants tend to gather at the top
or one side of the infected sesame plants,
giving the appearance of witch's broom.
Phytoplasma associated with sesame is
recognized as a serious risk for the cultivation
of sesame in several countries, including
Egypt, producing yield losses of up to 33.9
percent in yearly output (Abraham et al.,
1977). The phytoplasma are among the
obligate pathogens of plants and exist in the
phloem tissues of the infected host plants.
phytoplasma are being transmitted mainly by
insect vectors. The fact that phytoplasma
cannot be cultivated on artificial mediums and
can only be maintained in their plant hosts has
made the research of phytoplasma highly
arduous and complex. During the previous
years, PCR has been utilized for the
identification of a huge range of bacteria,
including phytoplasma. Several approaches
have been developed for the universal
detection of phytoplasma. The main difference
between these methods is the type of primers
used in PCR reactions. The primers used in
PCR to detect phytoplasma infecting plants are
normally designed to amplify a specific region
in highly conserved ribosomal (rDNA) genes.
Nested-PCR assays can increase both the
sensitivity and specificity of phytoplasma
detection in plant samples. The aims of this
study are to confirm the presence of
phytoplasma in symptomatic sesame plants
presenting evident phyllody symptoms by
employing polymerase chain reaction (PCR),
describe the main symptoms associated with
the disease in Assiut governorate, and evaluate
certain sesame varieties for their reaction to
phytoplasma infection under greenhouse
conditions. As well as the application of some
cobalt irradiation treatments on sesame seeds
to combat this destructive disease.
2. Materials and methods
2.1 Source of samples
The phytoplasma causal pathogen was isolated
from symptomatic sesame plants showing
typically symptoms of phytoplasma collected
from different fields in Assiut Governorate,
Egypt. All samples underwent DNA extraction,
molecular detection, and identification at the
Molecular Biology Unit, Assiut University,
Egypt. The DNA extraction was stored at -20°C
for subsequent tests.
2.2 Molecular identification of Phytoplasma
2.2.1 DNA extraction
The plant samples were disrupted using a T
mortar and pestle (100 mg wet weight or 20
mg lyophilized tissue). Subsequently, 400 ml
of Buffer AP1 and 4 ml of RNase A were
added to each sample. After Vortex, the
samples were incubated at 65°C for 10
minutes, and the tubes were rotated two or
three times. 130 ml of Buffer P3 were added to
each tube and incubated for five minutes.
Ahmed Hoda & Eraky Amal, 2022
34
2.2.2 PCR amplification, cloning and sequencing
Universal Primers for Detection of
Phytoplasma (S54LP of SP_F 50-C ATG GAG
GCC GAATTC ATG TTT AAA ATC AAA
AAT AAT TTA-30 and S54LP of SP_R 50-
GC AGGTCGACGGATCC TTA TTT TCA
TCA TTT AAA GTT TTT-30) (Maejima et al.,
2014) were used to confirm the presence of
Phytoplasma in symptomatic sesame plants.
The PCR outcomes were analyzed on a 1%
agarose gel, stained with ethidium bromide,
and observed under UV (Sambrook and
Russell, 2001).
2.3 Plants inoculation with phytoplasma
(Pathogenicity test)
Pathogenicity was carried out on sesame (Giza
32 cultivar). Sesame plant tissues exhibiting
characteristic phytoplasma symptoms were
collected and mashed in sterilized water with
the use of a pestle and mortar, and then pressed
through extremely fine muslin material.
Mechanically inoculated with the freshly
extracted sap using a syringe injection in the
stems of plants at different ages (4, 6, 8, and 10
weeks after planting), four pots were used for
each stage. Plants were washed with a mild
stream of water immediately after inoculation
to eliminate extra inoculum and put in insect-
free cages for symptom development. The
isolate proved its pathogenic capability in the
pathogenicity test, as it produced typical
symptoms of phytoplasma, including phyllody,
stunting, yellowing, and witch's broom
symptoms. Percentages of infection plants
were recorded at the end of the growing
season. The following equations were utilized
to calculate the disease incidence:
Percentage of infected plants = Number of
diseased plants / total number of plants × 100
2.4 Response of certain sesame cultivars to
phytoplasma infection
The response of three sesame cultivars (Giza
32, Shandawel 3, and Sohag 1) to phytoplasma
disease was evaluated under greenhouse
conditions by recording the incidence of
phytoplasma infection (incidence percentage
calculated on the basis of diseased plants over
the total plants assessed), as mentioned above.
Also, disease severity was recorded using a
scale of 0–6. Incidence and disease severity
were recorded according to Akhtar et al.
(2013), where 0 = no phytoplasma infection
(highly resistant), 1= 1–10 percent plant
infected (resistant), 2= 10.1–30 percent plant
infected (moderately resistant), 3= 30.1–50
percent plant infected (moderately
susceptible), 4= 50.1–75 percent plant infected
(susceptible), 5= more than 75.1 percent of
plants infected (highly susceptible). Disease
severity was converted into a percentage as
follows:
𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝑠𝑒𝑣𝑒𝑟𝑖𝑡𝑦 (%)=(0𝐴 + 1𝐵 + 2𝐶 + 3𝐷 + 4𝐸 + 5𝐹)
𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑔𝑟𝑎𝑑𝑒 𝑟𝑎𝑡𝑒 × 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 (5𝑇) × 100
Where A, B, C, D, E, and F are the number of
leaf plants corresponding to the numerical
grades 1, 2, 3, 4, and 5, respectively, and 8 is
the total number of plants multiplied by the
maximum disease grade (5).
2.5 Management of Phytoplasma associated
with sesame through irradiation
Four irradiation treatments (sesame seeds were
irradiated using Cobalt 60 at different doses of
150, 200, 250, and 300 Gy of gamma rays)
were evaluated under greenhouse conditions in
2022 for phytoplasma incidence.
2.5.1 Seed radiation treatments
A specified quantity (2 g per dosage) of dry,
homogeneous, and healthy seeds of Giza 32
cultivar of sesame were exposed to radiation
Ahmed Hoda & Eraky Amal, 2022
35
using a Co60 (Cobalt 60) gamma source with
varying dosages (150, 200, 250, and 300 Gy)
of gamma rays at Assiut University, Faculty of
Science, Department of Nuclear Physics. Five
pots were used as replicates, and each pot was
seeded with eight disinfected sesame seeds.
Sesame plants at age 8 weeks were challenged
with phytoplasma, and data on disease
incidence and disease severity were recorded
after 3 weeks of challenging sesame plants
with phytoplasma.
2.6 Statistical analysis
Data was analyzed to determine the relevance
of variations among treatments with regard to
phytoplasma disease severity. Once F-values
were significant (P<0.05), means were
compared using the least significant difference
(LSD) test (Gomez & Gomez, 1984).
3. Results and Discussion
3.1 Samples source
Sesame diseased and healthy samples were
obtained from Assuit Governorate, Egypt. All
collected samples were showing clear
symptoms of phytoplasma infection such as
phyllody, green leaf like floral, proliferation
and virescence except the healthy plant as
shown in Figure (1).
3.2 Symptoms of phytoplasma on sesame plants
Different forms of phytoplasma symptoms
were identified on sesame plants. phytoplasma
disease symptoms found in the samples
obtained from the field (Figure 1). The most
recognizable signs of the illness are the
transformation of floral parts into green leaf-
like structures, followed by extensive vein
cleaning in different floral sections. The ovary
is replaced with elongated structures, nearly
like a shoot. The calyx becomes polysepal,
while the sepals become leaf-like and remain
smaller in size. The phytoplasma flowers
become actiomorphic in symmetry, and the
corolla becomes polypetalous and deep green.
The veins of the bloom become thick and
fairly noticeable. The stamens keep their form
but become flattened, exhibiting a
predisposition to be leaf-like. The anthers turn
green and contain aberrant pollen grains. The
carpals are changed into a leaf fusion at the
borders, and this false ovary enlarges and
flattens, presenting a smooth feel and a
wrinkled surface due to the thickening of
capillary wall veins. Instead of ovules inside
the ovary, there are little petiole-like
outgrowths, which eventually expand and
burst through the walls of the false ovary,
giving small shoots (Figure 1). These branches
continue to develop and generate new leaves
and phytoplasma blooms. Plant diseases
caused by the presence of phytoplasma often
display a range of symptoms that are
suggestive of changes in the normal balance of
plant enzymes and hormones (Lee et al.,
2000). Also, Youssef Sahar et al. (2018)
observed that some symptoms in sesame are
like phytoplasma symptoms such as grouping
of branches of developing tissues, virescence,
which is pigmentation of non-green flower
parts to green, phyllody, formation of bunchy
fibrous secondary roots, weakness of plants,
reddening of leaves and stems, generalized
yellowing, and phloem necrosis. Jomantiene et
al. (1998) noticed similar symptoms in
strawberries. Recently, the availability of four
full-sequenced genomes has provided new
chances for the establishment of effective
control measures. As phytoplasmas lack the
cell wall, their membrane proteins and
released proteins work directly in the
cytoplasm of the host plants and insects. Thus,
the prediction of secreted proteins encoded in
the phytoplasma genome is vital for understanding
Ahmed Hoda & Eraky Amal, 2022
36
phytoplasma-host interactions. For example,
SAP11, SAP54, and TENGU have been
identified as phytoplasma effector proteins that
regulate plant gene expression, resulting in a
modification of plant morphology (MacLean
et al. 2011; Sugio et al., 2011).
A
B
C
D
Figure 1: Phyllody symptom of sesame. A= A shoot showing internodes shortening with dense leaves
(witches’ broom). B= yellowing sometimes accompanied the disease. C= The capsules turn into flowers.
D= Healthy sesame plant.
3.3 Molecular detection of sesame phytoplasma
strains
PCR amplification was used for phytoplasma
detection using the universal phytoplasma
PCR primer P1/P7 (Figure 2). Nested primer
R16F2n/R16R2 was used to confirm the
infection with a product size range of about
1250 bp for all infected samples. This
technique has been widely utilized for the
detection of phytoplasma and is probably the
most totally investigated. It recognizes all
strains of phytoplasma, whereas no findings
were obtained in health plants (Marzachi,
2004). The PCR approach readily separates
plant and phytoplasma so that a screening of
variations for the presence of phytoplasma
might take place (Schneider et al., 1995). PCR
has been widely used in the detection of many
organisms, including viruses. The results have
greatly contributed to determining the distribution
and diversity of the phytoplasma in sesame plants.
3.4 Pathogenicity test
Results in Table (1) showed that the age of
sesame plants affected the incidence of
phytoplasma diseases. Whereas injecting the
plants with phytoplasma after 8 weeks of
cultivation resulted in a high infection rate in
the plants with phytoplasma diseases.
Injecting the plants in the 6th and 4th weeks
gave a moderate phytoplasma infection. While
injections of plants in the 10th week of
cultivation gave the least percentage of
infection with phytoplasma.
Ahmed Hoda & Eraky Amal, 2022
37
Figure 2: Gel electrophoresis of PCR product amplified using universal
Phytoplamsa primers.
The findings revealed that the majority of the
plants were infected between 8 and 9 weeks
after seeding during the blooming season.
Sesame plants infected before blossom
initiation developed severe symptoms over the
entire plant and demonstrated full sterility.
This study, in accordance with Taye et al.
(2019), found that the degree of the change of
floral components into deformed structures
was connected with the period of infection.
However, plants infected during flowering
exhibited severe symptoms on the top of the
plants, occasionally followed by a few
rudimentary blooms that released extremely
small capsules with degenerated seeds. I also
observed that sickness. Incidence progress in
each treatment was a modest increase from 8
to 11 weeks after seeding. This study is in line
with Akhtar et al. (2009), who found that the
severity of the transformation of floral
components into deformed structures was
connected with the period of infection.
However, plants infected during blooming
exhibited severe symptoms on the upper part
of the plants, occasionally followed by a few
rudimentary blooms that delivered very little
capsules, and yellowing was also apparent.
The period of infection impacts the severity of
the phyllody illness. Infections in the early
stages of plant development had severe
symptoms on the whole plant, and plants
infected during blooming had severe
symptoms on the top portion of the plant.
Klein (1977) noted that plants infected early
displayed signs such as stunting, loss in leaf
size, and short internodes. It should be
emphasized that the increased prevalence of
the indirect symptoms in the research should
be driven by the late seeding of the nursery
beyond the regular sowing period.
Table 1: Infection percentage of sesame plants inoculated with phytoplasma.
Percentage of infection
30ab
40ab
60a
15b
36.13
3.5 Response of certain sesame cultivars to
phytoplasma infection
Data in Table (2) revealed that the lowest
percentage of infection and disease severity in
plants was observed in Sohag cultivars, as they
showed disease incidence and disease severity
of 7.5% and 2.5%, respectively, and could be
categorized as tolerant cultivars. whereas the
Giza 32 variety showed the highest percentage
Ahmed Hoda & Eraky Amal, 2022
38
of infected plants, about 40%, and also showed
the highest disease severity, so it could be
categorized as moderately susceptible.
Shandawel 3 cultivar also showed a small
degree of resistance to phytoplasma infection
as it developed disease incidence and disease
severity up to 10% and 18.50%, respectively.
These results indicate that sesame cultivars are
different in their response to phytoplasma
infection, and some sesame cultivars showed a
degree of resistance to phytoplasma infection.
Taye et al. (2019) discovered that sesame
plants infected before blossom initiation
exhibited severe symptoms on the entire plant
and indicated full sterility. A lot of writers
observed that the sesame crop was
substantially impacted by phytoplasma disease
in different regions (Singh et al., 2007; Win et
al., 2010; Mahmoud, 2013). According to
Akthar et al. (2013), from 133 sesame
genotypes that were tested for two years, there
were extremely significant variances in the
degree ofPhyllody disease resistance achieved
in the infection of all genotypes. Based on
infection percentage values, none of the
genotypes was classified as very resistant,
while 7 were resistant, 9 were moderately
resistant, 28 were tolerant, 33 were moderately
susceptible, 23 were susceptible, and 33 were
extremely vulnerable. The phytoplasma
disease has a significant effect on lowering
sesame output. The current investigation
demonstrated that the tested sesame varieties
have a distinct variation in the degree of
resistance detected between the varieties and
react differently (resistant, tolerant, and
susceptible) against Phyllody disease. Sohag1
variety was somewhat resistant, while Shandwel 3
variety is tolerant, but Giza 32 variety was
sensitive to phytoplasma illness. The use of
resistant cultivars is recognized as an affordable and
permanent technique for managing this
phytoplasma disease. Resistant, tolerant, or
immune plant varieties have so far been selected by
phytoplasma inoculation, symptom observation,
and variety selection (Jarausch et al., 2011), as
well as by marker-assisted selection programs
(Bisognin et al., 2009). Unfortunately, a
limited range of plant species have shown
resistance or tolerance to phytoplasmas.
Table 2: Response of certain sesame cultivars to phytoplasma infection under
greenhouse conditions.
Cultivar
Percentage of infection (%)
Disease severity (%)
Shandawel 3
10.0b
18.50ab
Sohag 1
7.50b
2.50b
Giza 32
40.0a
25.00a
L.S.D 5%
12.967
19.005
3.6 Management of phytoplasma associated
with sesame using cobalt seed irradiation
Four treatments by irradiation were applied to
manage the sesame phytoplasma under a
greenhouse in 2022. Data in Table (3) showed
all treatments by irradiation (150, 200, 250,
and 300 Gy) reduced phytoplasma infection.
Seed treatment with 300 Gy as sesame plants
treated with doase showed the lowest disease
incidence and disease severity (2.50 and
19.50) was more effective in affecting the
percentage of infected rate and disease
severity, respectively, with phytoplasma,
followed by 250 Gy (5.50 and 28.50) and 200
Gy (12.50 and 26.50), respectively. While
treatment with 150 Gy had the least effect by
rate (15 and 31.50), The study proved that
cobalt irradiation could be a useful method to
control phytoplasma associated with sesame
Ahmed Hoda & Eraky Amal, 2022
39
plants under Assiut governorate conditions,
and further studies are required to study the effect of cobalt irradiation under field
conditions.
Table 3: Effect of cobalt seed irradiation on sesame phytoplasma infection
under greenhouse.
Treatment
Infected plant (%)
Disease severity (%)
150 Gy
15.00b
31.50a
200 Gy
12.5b
26.50a
250 Gy
5.00c
28.50a
300 Gy
2.50c
19.50a
Control
40.0a
25.00b
L.S.D 5%
9.89
12.74
References
Abraham EV, Natarajan K, Murugesan M,
1977. Damage by pest and phyllody to
Sesamum indicum in relation to the time
of sowing. Madras Agricultural Journal
64(5): 298–301.
Akhtar KP, Sarwar G, Sarwar N, Elahi MT,
2013. Field evaluation of sesame
germplasm against sesame phyllody
disease. Pakistan Journal of Botany
45(3):1085–1090
Akhtar KP, Sarwar G, Dickinson M, Ahmad
M, Haq MA, Hameed S, Iqbal MJ, 2009.
Sesame phyllody disease: Symptomatology,
etiology and transmission in Pakistan.
Turkish Journal of Agriculture and
Forestry 33: 477–486.
Bertaccini A, Duduk B. 2009. Phytoplasmas
and Phtoplasma diseases: A review of
recent research. Phytopathologia Mediterranea
48: 355–378.
Bertaccini A, Duduk B, Paltrinieri S, Contaldo
N, 2014. Phytoplasmas and phytoplasma
diseases: A severe threat to agriculture.
American Journal of Plant Sciences 5:
1763-1788.
Gomez KA, Gomez AA, 1984. Statistical
procedures for agricultural research. 2nd
Edition, John Wiley and Sons, New York,
USA, p. 680.
Jomantiene R, Davis RE, Dally EL, Maas JL,
Postman JD, 1998. The distinctive
morphology of Fragaria multicipita'is due
to phytoplasma. HortScience 33(6):
1069–1072.
Jarausch W, Bisognin C, Schneider B, Grando
S, Velasco R, Seemüller E, 2011.
Breeding apple proliferation-resistant
rootstocks: Durability of resistance and
pomological evaluation. Bulletin of
Insectology 64: 275–276.
Klein M, 1977. Sesame phyllody in Israel.
Journal of Phytopathology 88(2): 165–171.
Lee I.M, Davis RE, Gundersen-Rindal DE,
2000. Phytoplasma: Phytopathogenic
mollicutes. Annual Reviews in Microbiology
54(1): 221–255.
Marzachì C, 2004. Molecular diagnosis of
phytoplasmas. Phytopathologia Mediterranea
43: 228–231.
Mahmoud MF, 2013. Induced plant resistance
as a pest management tactic on piercing
sucking insects of sesame crop.
Arthropods 2(3): 137–149
Maejima K, Oshima K, Namba S, 2014.
Exploring the phytoplasmas, plant
pathogenic bacteria. Journal of General
Plant Pathology 80: 210–221.
MacLean AM, Sugio A, Makarova OV,
Findlay KC, Grieve VM, Toth R,
Ahmed Hoda & Eraky Amal, 2022
40
Nicolaisen M, Hogenhout SA, 2011.
Phytoplasma effector SAP54 induces
indeterminate leaf-like flower development in
Arabidopsis plants. Plant Physiology 157:
831–841.
Schneider B, Cousin MT, Klinkong S,
Seemüller E, 1995. Taxonomic
relatedness and phylogenetic positions of
phytoplasmas associated with diseases of
faba bean, sunnhemp, sesame, soybean,
and eggplant. Journal of Plant Diseases
and Protection 102: 225–232.
Sambrook J, Russell DW, 2001. Molecular
cloning: A laboratory manual, 3rd edn.,
Cold Spring Harbor Laboratory Press,
New York, USA.
Singh PK, Akram M, Vajpeyi M, Srivastava
RL, Kumar K, Naresh R, 2007. Screening
and development of resistant sesame
varieties against phytoplasma. Bulletin of
Insectology 60(2): 303–304.
Sugio A, Kingdom HN, Maclean AM, Grieve
VM, Hogenhout SA, 2011. Phytoplasma
protein effector SAP11 enhances insect
vector reproduction by manipulating
plant development and defense hormone
biosynthesis. Proceedings of the National
Academy of Sciences of the United States
of America 108: 1254–1263.
Taye W, Alemu Z, Getahun S, Abate S, Seid
N, Hemba M, 2019. Evaluation of sesame
varieties for phyllody disease resistance.
Academic Research Journal of
Agricultural Science and Research 7(3):
110–118.
Win NKK, Back CG, Jung HY, 2010.
Phyllody phytoplasma infecting sesame
(Sesamum indicum) in Myanmar.
Tropical Plant Pathology 35(5): 310–313.
Youssef Sahar A, Safwat G, Shalaby ABA,
El-Beltagi HS, 2018. Effect of
phytoplasma infection on plant hormones,
enzymes and their role in infected sesame.
Fresenius Environmental Bulletin 27(8):
5727–5735.