الفهرس 
Studies on mixed infection of viruses and phytoplasmas in naturally infected Ocimum basilicum l. (basil) in Egypt
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Abstract
Sweet basil (Ocimum basilicum L.) is cultivated in Egypt as an important ornamental,
culinary, and medical herb. It was found to be naturally infected in the campus of Cairo
University, El-Giza governorate. The infection induced disease symptoms, including
mosaic mottling, leaf distortion, blistering, witches’ broom, and phyllody. Biological,
serological, and molecular studies proved that the infection is due to Tobacco mosaic
virus (TMV) and phytoplasmas. The effect of this infection on the histology, cytology,
and essential oil composition of the plant was also studied.
Part I: Pathological changes in sweet basil (Ocimum basilicum L.) caused by mixed
infection of tobacco mosaic virus and phytoplasmas.
To confirm the identification of TMV and phytoplasmas; host range, serology (DASELISA),
molecular (PCR and nested PCR), light and transmission electron microscopy
were done. Histopathological and cytopathological changes were done to study the
effect of the mixed infections in plant tissues and cells.
The results showed that the virus could be mechanically transmitted, exerting primary
symptoms. It infected Nicotiana tabacum, Datura metel, and Datura stramonium
forming chlorotic local lesions, and it was transmitted to Phaseolus vulgaris showing
necrotic local lesions.
DAS-ELISA revealed positive TMV infection during summer (July, August, and
September) recording O.D. 0.541, 0.794, and 0.239, respectively. While during the rest
of the year TMV concentration decreased. These results indicated that TMV
concentration was highly affected by climatic changes. The virus infection was identified using molecular techniques. Total RNA was extracted
from infected and control leaves using RNeasy plant Mini Kit then reverse transcribed
with Revert Aid First Strand cDNA Synthesis Kit. The produced cDNA was tested for Alfalfa mosaic virus (AMV), Chilli veinal mottle virus (ChiVMV), Pepino mosaic virus
(PepMV) and TMV. All the above-mentioned viruses were not detected in the sample
except for TMV that generated a band of ≈500bp. The produced DNA was subjected to
semi-nested PCR to give a specific band at ≈500bp suggesting the infection by TMV.
Transmission electron microscopy of purified virus showed rod shaped particles
measuring " ~ "300 nm in length and " ~ "26 nm in width confirming the infection of basil
with TMV.
To test for the presence of phytoplasma; total DNA was extracted from leaf midribs of
the infected sweet basil using EZ-10 Spin Column Plant Genomic DNA Miniprep Kit
and tested by PCR using primer pair P1/P7 and nested PCR using specific primers of
phytoplasma (R16F2n/R16R2) that generated a DNA band at the expected size (≈1200
bp).
Further Candidatus phytoplasma identification was done using sec genes. PCR was
done using primer pair (SecAfor1/SecARev3) producing a specific band at ≈ 840 bp.
The PCR product was then diluted and used as template for semi-nested PCR using the
primer pair (SecAfor1/SecARev2) which generated a band at ≈700 bp suggesting that
the isolated Candidatus phytoplasma from sweet basil belong to major 16Sr groups
including 16SrII and excluding 16SrXI phytoplasmas since the later cannot be detected
using secA genes
For further biological investigation for the presence of phytoplasma; thin sections of
infected basil stem showing necrotic streaks were subjected to Dienes’ stain, and
examined using light microscopy. Blue color pleomorphic bodies were observed in the
phloem tissues indicating the presence of phytoplasma.Histopathological and cytopathological studies were done on infected basil leaves to
study the alterations associated with the mixed infection
Histopathology of infected leaves showed hypoplasia in the yellow mottled areas, and
hyperplasia green vein banding. The lamina was thinner in the yellow areas when
compared to the dark green areas, and the mesophyll cells were less differentiated.
Blisters appeared as a convex and concave curvature, with asymmetric thickness of the
mesophyll. Leaf blade showed a downward bending. Reduced vascular tissues with a
low number of phloem cells and xylem vessels were observed in leaves infected with
virus only while leaves infected with mixed infection of virus and phytoplasmas showed
excessive vascular tissues.
Transmission electron micrography showed that the mixed infection caused
degeneration and deformation of cell organelles in contrast to control. The cell wall was
thick with abnormal protrusion to the inside of the cell.
Ultrastructural electron microscopy revealed the presence of inclusion bodies of TMV
in the cytoplasm in the form of hexagonal crystals and paracrystalline, fibrous, spindleshaped
bodies. Also, the phtyoplasma pleomorphic, wall-less structure appeared.Part II: Phytochemical composition of essential oils from aerial parts of sweet basil
infected by mixed infection of tobacco mosaic virus and phytoplasma
Infected sweet basil was subjected to Gas chromatography- Mass spectrophotometry
(GC-MS) analysis to study the changes that may occur in the essential oil composition.
The analysis was done on TMV infected, phytoplasma infected, mixed infection of
(TMV & phytoplasma) and healthy (not infected) leaves of sweet basil.
Hydrodistillation at Clevenger-type apparatus was done for healthy and infected
samples. The essential oil was recovered then dehydrated separately with anhydrous
sodium sulphate.
The GC-MS analysis of the essential oil samples was carried out using GC-MS
instrument stands at the Department of Medicinal and Aromatic Plants Research,
National Research Centre. The compounds were identified by calculat ing their retention
index (RI) relative to alkanes (C8-C22) then compared with those reported in the
literature. The recorded mass spectra were compared to those contained in Wiley
spectral library collection and the National Institute of Standards and Technology
(NIST) mass spectral library.
Thirty different essential oils were extracted. where the yield found to be
(0.055±0.01%) for the control, (0.143±0.02%) for the mixed infection of TMV and Candidatus phytoplasma, (0.13±0.02%) for TMV infection and (0.22±0.04%) for
Candidatus phytoplasma infection. The essential oil composition was severely altered.
Monoterpenes hydrocarbons showed a significant increase in the mixed infection plants
(5.87±0.77%) than those infected by a single infection of either TMV (2.55±0.19%) or
Candidatus phytoplasma (2.42±0.18%) when compared to the uninfected control
(2.22±0.33%). The oxygenated monoterpenes showed a high significant increase in the
mixed infection (72.76±0.05%) followed by a significant increase in Candidatus
phytoplasma infection (55.43±4.63%) while there was no significant increase in case of
TMV infection (48.47±3.68%) compared to the control (49.12±2.24%). Ketones
increased significantly in all infections, showing the highest significance in the mixed
infection (8.9±0.3%), followed by TMV infection (5.69±0.36%). The least significance
was recorded in Candidatus phytoplasma infection (5.02±0.38%) compared to the
control (0.45±0.03%). However, sesquiterpenes hydrocarbons and oxygenated
sesquiterpenes showed a significant decrease in the infected plants. The highest
significant decrease was recorded in Candidatus phytoplasma infection (5.34±0.37%)
followed by TMV infection (7±0.048%). A significant decrease in sesquiterpenes
hydrocarbons was recorded in the mixed infection (8.74±0.05%) compared to the
control (26.84±0.16%). The oxygenated sesquiterpenes significantly decreased in the
mixed infection (3.27±0.02%) compared to the control (17.33±0.07%) and were not
determined neither in the TMV nor in the Candidatus phytoplasma infections which
confirmed that the mixed infection severely changed sweet basil chemotype.The significant increase of some essential oils is suggested to be attributed to defense
mechanism, plant–insect interactions, or pollination. However, the decrease of some
essential oils such as Linalool and β-ocimene may be due to the malformation of leaf.
from the previous findings, we can provide evidence for the co-existence of
TMV and phytoplasmas in sweet basil. The presence of both pathogens was
correlated with climatic changes. They cause an important alteration in sweetbasil tissue and essential oils composition. Therefore, they have direct or indirect
metabolic consequences in commercially important medicinal plant species such
as sweet basil.
Further studies are recommended to understand the synergism between the virus
and phytoplasma in mixed infection under natural conditions, the mechanisms by
which these pathogens are transmitted together to the same plant host, their extent
throughout the country, and the factors determining their coexistence, in addition
to any epidemiological implications and impacts that may occur from geographic
location and/or climate conditions.