UNIVERSITY OF AGRICULTURE, FAISALABAD

Centre of Agricultural Biochemistry and Biotechnology (CABB)

Faculty of Agriculture
(SYNOPSIS FOR M.PHIL BIOTECHNOLOGY)
TITLE: INDUCTION OF LEAF CURL DISEASE IN CHILLIES USING
DIFFERENT MONOPARTITE AND BIPARTITE BEGOMOVIRUSES

Name of the Student Hina Fatima

Registration Number 2022-ag-2325

Abstract
Chilli (Capsicum annum) is one of the most significant economic crop in Pakistan and
worldwide. In spite ofaddition to being used as food, it is also used as an essential flavouring
spice in regional cuisines. Viruses are the root cause of abundant many plant diseases that reduce
crop quality and productivity. Chilli plants are infected by many viruses, especially
begomoviruses, a member of the Geminiviridae family, showing curling of leaves and stunted
growth as symptoms. In this study, the agroinfiltration will be used to inoculate infectious clones
of different begomoviruses in chillies. For this purpose, the inoculum of different begomoviruses
will be prepared and agroinfiltered in chilli leaves at the three leaves stage. After 21 days,
symptoms will be observed in systemic leaves and then total DNA will be extracted from
systemic leaves following the onset of symptoms and analyzed by PCR analysis for confirmation
of the movement of virus. This research will demonstrate great promises to improve the field of
study as it relates to the interaction between plant and virus, advance disease management
methods, and promote the sustainability and production of chilli.
 UNIVERSITY OF AGRICULTURE, FAISALABAD
Centre of Agricultural Biochemistry and Biotechnology (CABB)
Faculty of Agriculture
(SYNOPSIS FOR M.PHIL BIOTECHNOLOGY)
I. TITLE: INDUCTION OF LEAF CURL DISEASE IN CHILLIES
USING MONOPARTITE AND BIPARTITE BEGOMOVIRUSES

II. a) Date of Admission 26-09-2022

b) Date of Initiation 20-09-2023
c) Probable Duration 1 year (2 Semesters)

III. PERSONNEL
a) Name of the Student Hina Fatima
b) Registration Number 2022-ag-2325

IV. SUPERVISORY COMMITTEE

i. Supervisor: Dr. Muhammad Mubin ([email protected])
ii. Member: Dr. M. Shah Nawaz-ul-Rehman ([email protected])
iii. Member: Dr. Ameer Bibi ([email protected])

V. Need for the Project

Chilli (Capsicum annuum) belongs to family Solanaceae and genus capsicum (Padhi et al.,
2017). There are twenty species in genus Capsicum, out of them five are classified as domestic
varieties. It can grow in tropical and subtropical areas (Islam et al., 2018). Originates from
South and Central America and is cultivated everywhere almost in the world. Portuguese brought
it to the Indian subcontinent in the 17th century, and it quickly became the part of the national
cuisine (Sathiyamurthy et al., 2019). Vitamin A, L-ascorbic acid, Vitamin B6, Potassium, Iron,
and Magnesium are among the nutrients which are found in chillies (Vysali et al., 2021). Chilli
fruit comprises an essential oil named oleoresin which is utilized to make value-added products
in the food, beverage and pharmaceutical industries (Mishra et al., 2020). The compound
capsaicin found in it is used as a medication to treat a variety of conditions in humans, including
non-allergic rhinitis, neuralgia, and rheumatic disorders (Bhutia et al., 2018). Globally, 18560
square meter of land is used for growing chillies, producing 4.626 million tons of crop product
(Rais et al., 2021). Pakistan ranks fourth globally in terms of production of red chillies. In
Pakistan, the area under chilli cultivation is 65,300 hectares, and the quantity of chili produced in
2018–19 is 148.7 thousand tonnes, an increase from 148.1 thousand tonnes in 2017–18 (Channa
et al., 2020). Pathogens, especially bacteria, viruses, nematodes and fungi, pose a significant
threat to the chilli crop. In chilli plants, fungus cause different infections like Phytophthora
blight, damping off, and Fusarium wilt. The crop of chillies is also affected by bacteria. Bacterial
wilt, leaf spot, bacterial canker, syringe seedling blight and bacterial spot are a few prevalent
bacterial diseases (Hussain et al., 2011). Chilli is vulnerable to several contagions; including
viruses, which can result in significant losses in yield. Leaf curl disease has been linked with
four begomovirus species in India and Pakistan (Thakur et al., 2018). Geminiviruses that contain
single-stranded DNA cause serious destruction to food crops around the world. Within the family
Geminiviridae, the most significant group of plant viruses is found in the genus Begomovirus
(Akhter et al., 2014). Begomoviruses only infect dicots and are spread by whiteflies (Bemisia
tabaci). These are classified as either monopartite (having a 2.8 kb genome with six ORFs) and
bipartite (having DNA-B that is the same size as DNA-A). DNA-A enables replication and
transcription functions while DNA-B provides movement functions (Nawaz-ul-Rehman et al.,
2009). Currently, around seventy-five viruses are known to infect chilli. In recent times, Chilli
leaf curl disease has become a major problem, which considerably lowers commercial yield of
these crops (Shingote et al., 2022). Symptoms of ChiLCD infection include (curling reduced leaf
size, puckering and rolling of leaves). In severe situations, plants become stunted and are unable
to produce fruit, which results in the harvest being lost entirely (Jeyaraj et al., 2023). This
research intends to induce various leaf curl diseases in chilli plants using the agroinfiltration
method, aiming to assess the impact of these diseases on plants. The inoculum containing
infectious molecules of different begomoviruses will be prepared and subsequently injected into
chilli leaves using the syringe, enabling the transient expression of numerous leaf curl viruses.
 Objectives

 To investigate the efficiency of Agroinfiltration in chillies

 To evaluate the consequence of co-infection with multiple leaf curl viruses on chilli
plants

VI. Review of Literature

Agroinfiltration is the utmost important method to check the utility of genomic components of
viruses. Mung bean yellow mosaic virus (source of Yellow mosaic disease) in Mung bean and
MYMIV caused a serious threat to overall Mung bean production. Utilizing infectious Agro-
constructs of Mung bean yellow mosaic India virus (DNA-A and DNA-B) an effective and
repeatable agroinfiltration approach was used to obtain maximal efficiency. The current study
objective was to look at the optimal tissue types for maximum infection by damaging a variety of
meristematic tissues. The inoculation on the epicotyl area exhibited the highest level of
infectiousness among the different tissues chosen. Additionally, standardization of
acetosyringone concentrations, incubation times and Agrobacterium cell density was done to
increase MYMIV's infectivity. The highest infection was observed on the first ternate leaf during
ten to twelve days after the damaged sprouted seeds were sown in soil and incubated for 2-4
hours in one optical density of culture carrying a repeat construct of MYMIV without
acetosyringone (Sivalingam et al., 2022).

In cotton fiber, a simple and quick Agrobacterium-mediated technique was established to

temporarily transform the genes and promoter. Using GUS and GFP as reporters, first discovered
that exogenous genes were expressed in it. Subsequently, the LBA4404 Agrobacterium strain,
the three-hour infection period, and the two-day incubation period were identified as
characteristics that influence transformation efficiency. Transiently transforming 4 distinct
genes (cotton)each of which are found exclusively in fibers, were shown to be temporarily
altered in cotton fibers being found 10-1000 times higher than those of the control. Beta
glucuronidase staining and activity analysis showed that the GhMYB212 and GhFSN1
promoters in transformed fibers have activity profiles that are comparable to their original
activity in developing fibers. It has been determined that the temporary transformation approach
is appropriate for subcellular localization research (Shang et al., 2022).

Rumex nepalensis, which is grown in Himachal Pradesh, India, shown a symptoms which are
begomovirus like during a survey conducted in 2013. They were identified as the (cotton leaf
curl Multan betasatellite and tomato leaf curl Palampur virus) via complete sequence
characterization. Clones of (virus and betasatellite) are agro-infiltrated in R.nepalensis and
Nicotiana benthamiana. Plants infected with DNA-A alone failed to display a disease-like
phenotype. However, significant symptoms were noted when combined with CLCuMuB. In all
the aforementioned combinations, agro-infiltrated R. nepalensis plants weren't showing any
outward signs. The presence of these (Tomato leaf curl Palampur virus and cotton leaf curl
Multan Betasatellite) in plants were confirmed by PCR and Southern blotting. Bipartite
ToLCPalV linked to CLCuMuB has a new natural host, identified as R. nepalensis (Sharma et
al., 2019).

The advancement of transient gene expression in plant biotechnology is due to the development
and enhancement of powerful transformational tools. This study investigated the intrinsic
resistance against ToLCNDV in tomato leaves by introducing a viral clone via agroinfiltration
which is a quick, comparatively cheap, and efficient technique. This is a novel study since a PCR
reaction verified that the viral genome was successfully transferred to Agrobacterium
tumefaciens using vacuum infiltration. Money Maker and Tomato F1 pound are two of the five
locally grown tomato cultivars that are vulnerable to ToLCNDV, with the other three being
naturally resistant. Additionally, these vulnerable cultivars can be employed in breeding
programs to build resistance and boost economic yield (Ali et al., 2023).

Capsicum annuum is a significant vegetable and spice. Using hypocotyl explants and an effective
Agrobacterium Facilitated process, develop a proficient and repeatable IAA free regeneration
procedure for 6 distinct varieties. Six different red pepper cultivars axenic seedlings were used to
harvest explants. The explants were then cultivated on MS media, which contains 6-
Benzylaminopurine/ in conjunction with Indole-3-acetic acid. 2 varieties, LCA-235 and Supper,
exhibited increased elongation when gibberellic acid added to the shoot elongation media at a
con. of 0.5 mg/l, although no discernible change was observed in the other cultivars.
Agrobacterium tumefaciens was used to transform the chilli cultivars. Southern hybridization
 studies together with PCR were used to confirm transgenic incorporation in putative
transformants (Kumar et al., 2012).

A competent transforming procedure for the 2 best of chilli (Pusa Sadabahar and Pusa Jwala)
was performed. To facilitate effective transformation in these cultivars, using cotyledonary and
hypocotyl explants, made an effective and repeatable Agrobacterium-facilitated transformation
process for chilli. The hypocotyl and cotyledon explants were regenerated Eighty-one percent in
Pusa Sadabahar and seventy-eight percent in Pusa Jwala using the optimum regeneration
medium (have 1 mg l-1 IAA and 5 mg l1 6-BAP). Agrobacterium tumefaciens strain LBA4404,
which carries the pCAMBIA 2301 construct containing the beta-glucuronidase reporter gene and
NPT-II marker genes, was used to standardize the transformation procedure. Their work showed
that the best materials for genetically engineering chillies using an Agrobacterium based method
are explants made from the hypocotyls of young seedlings. Agrobacterium cells were discovered
to be the best cultivars to co-cultivate hypocotyl explants for a length of seventy-two hours to
achieve a tremendous transformation efficacy of roughly thirty present in both cultivars (Mahto
et al., 2018).

VII. Materials and Methods

Sample collection and Research site:
Chilli seeds will be collected from the Ayub Agricultural Research Institute, Faisalabad and
seeds will be grown under controlled conditions in virology lab, CABB, University of
Agriculture, Faisalabad.
Growth condition for plants:
Chilli plants will be grown in the growth room of the virology Lab on peat moss at 24-28 C℃
and 16 hours of daylight.
Infectious Clone Transformation
Infectious clones of viruses (Tomato leaf curl New Delhi virusToLCNDV, Cotton Leaf Curl
Multan virus, and Paper Leaf Curl Bangladesh virus) will be transformed in competent cells of
Agrobacterium strain GV3101. L.B. agar plates that containing kanamycin, gentamycin and
rifampicin will be used to culture colonies of these transformed agrobacteria. ColoniesThis
culture will be added to L.B. broth, and the mixture will be kept in a shaking incubator overnight
at twenty-eight degrees. Chilli plants will be inoculated with this culture at the three-leaves stage
and will most likely show symptoms after 21 days.
Screening of plants for infection
The presence of infection will be examined by noticing disease symptoms after twenty-one days
of infiltration. DNA from symptomatic and non-symptomatic leaves will be extracted by the
CTAB method and replication of begomoviruses will be confirmed by PCR. Then inveterate the
PCR product by running gel electrophoresis.
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