الفهرس 
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Abstract
PVY is a serious pathogen that infect several important crop species in the solanaceae family including potato, tobacco, pepper, and tomato. Infection with PVY spread easily and the yield losses reached to 80%.PVY is a member of Potyviridae, the largest plant virus family. It is found worldwide wherever potatoes are grown.
Potatoes are Egypt’s most important export vegetable crop and the second most important (after tomatoes) vegetable crop in economic value. However, potato plants are usually subjected to numerous diseases caused by viruses, fungi, bacteria, phytoplasma and viroid. Viral diseases are mainly responsible for the majority of losses caused in potato production especially Potato virus Y (PVY).
This work was carried out at Virology Lab. and greenhouse of Department of Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt from 2005 to 2007. The present study aimed to biological, molecular characterization on the three isolates of Potato virus Y as a common virus in Egypt and a dangerous pathogen of potato plants.
Obtained results could be summarized in the following points:
I. Confirmation of PVY isolates.
A. Indicator plants.
PVY-isolates gave different morphological local lesions (shape, diameter, center and size) on Ch. amaranticolor L. about 15 days post inoculation. N. rustica L .showed chlorotic spots and mosaic about 15 days post inoculation with the three PVY-isolates (A, B and C).
B. DAS-ELISA.
The three isolate of PVY which show different symptoms on Ch. amaranticolor L .and N. rustica L. were confirmed by using DAS-ELISA whereas, gave positive serological reaction with PVY-specific polyclonal antibodies by different values at 405 nm.
II. Propagation of PVY isolates.
The three isolates of PVY (PVY-A, PVY-B and PVY-C) were propagated on systemic host N. tabacum cv. Samsun NN.
III. Differentiation between the three PVY-isolates
1. Biologically:
1.1 Differential hosts.
PVY-isolates could be distinguished according to the different degrees of symptoms on hosts, and the produced symptoms were divided into two categories:
Localized symptoms:
The three isolate of PVY (A,B,C) gave different morphological chlorotic local lesions on Ch. amaranticolor L.,. And N. rustica L., showed chlorotic spots and mosaic on the inoculated leaves.
Systemic symptoms :
The three isolates of PVY were inoculated on healthy host plants C. annuum cv. California wonder, D. metel L., L. esculantum L. cvs. Castle rock, Ph. floridana, Nicondra physaloides L., N. glutinosa L., N. rustica L., N. tabacum cv. Samsun NN. The inoculated plants showed different systemic symptoms. L. esculantum L. cvs. Castle rock, were produced variable symptoms with PVY isolates; veinal necrosis and epinasity with PVY-A isolate but it were produced leaf-cup shape, crinkle, leaf narrowing, leaf malformation and small leaflets with PVY-B isolate but in case of PVY-C isolate they were produced severe mosaic, leaf deformation and crinkle.
1.2 Mode of transmission:
1.2.1 Mechanical transmission(sap transmission):
PVY-isolates (A,B&C) were mechanically transmitted by easily to the potato cvs., and differential hosts by infectious crude sap of each PVY-isolate.
1.2.2 Aphid transmission:
The results showed that, PVY-isolates were non-persistent manner transmitted by Myzus persicae Sulz from infected potato cv. Diamont plants to healthy ones. The inoculated plants showed PVY symptoms.
1.3 In vitro properties of PVY-isolates:
The three PVY-isolates were differed in their stability in infectious crude sap of D. metel L., leaves which TIP was 64, 66 and 60 C for PVY-A, PVY-B and PVY-C respectively. DEP was 10-5, 10-4 and 10-3 for PVY-A, PVY-B and PVY-C respectively and also LIV was 4, 6 and 4 days for PVY-A, PVY-B and PVY-C respectively.
1.4 Light microscope examination:
1.4.1 Epidermal cells and stomata appearance
The epidermal cells of PVY-isolates infected leaves were appeared shrinked with destructed cell walls. The three PVY-isolates affects on the stomata shape in infected leaves, these stomata were appeared closed due to the lignifications in the stomata gap.
1.4.2 Inclusion bodies
The three PVY-isolates appeared differences in formation of cytoplasmic inclusions whereas the numbers of crystalline inclusions were differed for PVY-A, PVY-B and PVY-C. As well as, the three isolates of PVY induced the different numbers, shape and size of amorphous inclusion bodies.
1.5 Susceptibility of potato cultivars to infection with the three PVY-isolates
1.5.1 Disease symptoms
Potato cultivars Lady Roseatta, Herms, Draga, Cara, Burren, Diamont, Spunta, and Nikola were inoculated by three PVY-isolates which were gave the following symptoms:
PVY-A isolate produced vein clearing and mosaic on Lady Roseatta, yellowing on Herms, Draga and Spunta, necrosis on Cara and apical necrosis on Burren, Diamont and Nikola.
PVY-B isolate showing vein clearing on Lady Roseatta, yellowing on Herms, Cara, Diamont, Spunta and Nikola. But Draga cultivar gave necrosis and Burren gave apical necrosis.
PVY-C isolate gave yellowing on Herms, Draga and Cara, however, necrosis was noticed on the Burren. And apical necrosis was showed on Nikola. But no symptoms were recorded on Lady Roseatta, Diamont and Spunta.
The flowers of potato cv. Diamont were appeared some of symptoms, whereas PVY-A, PVY-B isolates gave necrosis, petals color breakdown and PVY-C produced abortion of flowers.
1.5.2 Disease severity values of infected potato cvs.
Potato cultivars which were inoculated with PVY-isolates gave different disease severity values, whereas, Spunta gave a lowest disease severity which were recorded 20% DS with PVY-A, PVY-B isolates while 30% DS with PVY-C isolate.
Burren and Diamont were produced a highest disease severity which were recorded 100% DS with PVY-A. And they produced 100%, 40% DS with PVY-B isolates respectively while 20%,66.6% DS with PVY-C isolate.
1.5.3 Serological detection of PVY-isolates:
1.5.3.1 DAS-ELISA
The three PVY-isolates (A, B and C) were positively reacted with the PVY-specific polyclonal antibodies. The difference in the serological reaction between virus isolates were determined by DAS-ELISA test.
2. Serological relationship among the three PVY-isolates.
2.1 Double diffusion test in plates:
The three PVY-isolates gave positive precipitation reaction with specific PVY-IgG and a visible line of precipitation was differed for each isolate. Whereas, PVY-B isolate gave strong reaction while PVY-A, PVY-C gave faint reaction.
2.2 Monoclonal antibodies (Serotype)
TAS-ELISA, indicated that PVY-A and PVY-B isolates are belonging to PVYO and PVYC while PVY-C isolate was belong to PVYN –strain.
3. Molecular variabilities among PVY-isolates.
3.1 Reverse transcription-polymearase chain reaction. (RT-PCR)
PCR reaction appeared differences between PVY-isolates based on number and sequence of base pairs of coat protein gene ; as well as height and area of PCR peak. Whereas, PVY-A gave (859 bp), PVY-B produced (872 bp), and the number of base pairs of coat protein gene of PVY-C isolate was (839 bp).
3.2 Nucleic acid spot hybridization (NASH):
Dot blot hybridization was used to confirm and compare between the three PVY- isolates (A,B and C).
It was found that PVY-A, PVY-B and PVY-C isolates were closely related with comparison by color, density and diameter. Whereas, PVY-A isolate appeared pale purple color and spot diameter 0.4 cm, PVY-B gave intense purple colour and 0.45 cm spot diameter and as well as PVY-C produced purple color and spot diameter 0.45 cm.
IV. Interaction between PVYN-strain and potato cv. Diamont.
1. Physiological changes in PVYN-infected potato plants cv. Diamont.
1.1 Changes in Enzyme activities:
The level of peroxidase, polyphenoloxidase are found to be considerably higher in infected leaves than healthy ones. The relative activities of these enzymes were higher in potato plants (cv. Diamont) infected with PVYN-strain 60.49 and 33.33 % for peroxidase and polyphenoloxidase respectively than healthy ones.
The levels of catalase and amylase were reduced in infected plants than healthy ones. The relative activities of these enzymes were lower in infected potato leaves 39.18 and 5.48 % for catalase and amylase respectively than healthy ones.
1.2 Changes in photosynthetic pigments:
Chlorophyll a and chlorophyll b were decreased in PVYN-infected potato leaves than healthy ones while carotenoides levels were higher in infected leaves than healthy ones. The reduction percentage of total pigments in infected leaves was 39.89 %
1.3 Changes in total carbohydrate content in potato leaves and tubers:
Total carbohydrate content differs greatly in leaves infected with PVYN-strain and healthy ones and the reduction percentage of total carbohydrates content in PVYN-infected potato leaves were 21.74%.
Total carbohydrate content and the total count of starch granules were reduced greatly in potato tubers infected with PVYN-strain than healthy ones. the reduction percentage of total carbohydrates content in PVYN-infected potato tubers were 40.19%.
The numbers of starch granules from healthy potato tubers gave 4.43 X 105 while from PVYN-infected potato tubers were recorded 2.83 X 105 / ml of tuber suspension, and the percentage of reduction was 36.11 % and also the PVYN-infected tubers showed distorted starch granules.
1.4 Changes in total nitrogen content and dry matter.
The rate of reduction in nitrogen content of PVYN-infected potato leaves compared with healthy ones were 18.09 %.
The reduction in dry matter content of PVYN-infected potato leaves was2.97 %.
1.5 Changes in total protein
The rate of reduction in total protein of PVYN-infected potato leaves compared with healthy ones were 18.07 %.
1.6 Changes in nucleic acids ( Total RNA)
The concentration of RNA in infected leaves was significantly higher than healthy leaves. It was found 208.2 and 168.1 ug/ml for infected and healthy leaves respectively and the rate of increases was 23.85 %
2. Cytopatheic effects:
2.1 Histopathological effects:
Light microscopy revealed several changes in both petiole and blade of potato cv Diamont leaves due to PVYN-infection. PVYN- infected potato leaf blade showed destructed upper epidermis cells due to necrosis, hypertrophy of parenchyma cells of spongy tissue and deformation of palisade cells. The phloem tissue contained much less active sieve elements and the xylem vessels attained less diameter as compared with those healthy ones. The percentage of reduction in the area of vascular cylinder for infected potato leaf petiole was 28 % Consequently, the vascular cylinder dimensions were lessened than healthy ones.
2.2 Ultrastructure studies:
The ultrathin sectioning technique using transmission electron microscope for examining ultra structure of the potato leaves which were infected with potato virus Y (PVYN-isolate), as well as healthy leaves.
The PVYN-infected potato leaves cells showing:
• Cytoplasmic inclusion formed by PVYN, they may contain virus particles, and also observed by light microscopy.
• Changes in chloroplastids like clumped, partially destructed with reduction in size and number. And abnormal appearance of grana, thylakoid membrane.
• The abnormal appearance of mitochondria and nucleus. Elongated and curved mitochondria was detected in infected cells, nucleus with several dark stained bodies.
As well as PVYN-infection destruct the vascular bundles of potato plants cv Diamont, whereas, the xylem vessels attained less diameter than healthy ones and contained several spherical dark stained-bodies which were precipitated on vessels walls. A little sieve tubes were detected with destructed companion cells. These changes in vascular bundles retards their growth and may be lead to death of potato plants.
V. Molecular characterization of PVYN-strain:
1. RT-PCR:
RT-PCR of the PVY/CP-Egyptian isolate (N) was used to amplify a fragments of about (839 bp).
2. Nucleotide sequence analysis:
The partial nucleotide sequence of the PCR-amplified fragment for the coat protein (CP) gene of PVYN-strian was done to determine the relationship with other recommended PVY strains registered in GeneBank.
The cDNA sequence was performed using PCR produced when the specific (downstream and upstream) primers for CP gene of PVY were used. Nucleotide were found to be 351 bp from CP genome sequence. Codon start with AGA, A phylogenetic tree of PVY-EG revealed 42% a moderate degree of similarity to the other PVY isolates sequences.
The difference in G/C, A/T ratio between the Egyptian PVYN- strain and two PVY strains, PVY-8084NTN (Accession no. EF027897), PVY-H (Accession no. X54611) were detected whereas, the Egyptian PVYN-strain was recorded 1.16, 1.37 and the two PVY strains were recorded 1.27, 1.32 for G/C, A/T ratio respectively.
3. Translation of PVYN-strain partial (CP) gene nucleotide sequence:
The partial nucleotide sequence of the coat protein gene was translated into its deduced amino acids.
The amino acids composition of partial CP gene sequence for PVY-Egypt (Accession no. EF502038) and two PVY-isolates published in GeneBank, PVY-8084NTN (Accession no. EF027897) PVY-H (Accession no. X54611) were 116 AA.
4. Nucleic acid spot hybridization (NASH).
The prepared PVY/CP DNA probe was used in dot blot hybridization technique in order to test for the presence of PVYN. The probe successfully hybridized with samples of PVYN-infected D. metel. L leaves which produced purple color and spot diameter 0.45 cm.
Table (20): Characterization of PVY-strains
Characters PVY-A PVY-B PVY-C
Biological characterization
Differential hosts
Ch. amaranticolor L. Small, circular chlorotic local lesions Numerous, large chlorotic local lesions Numerous, irregular chlorotic local lesions
Ph. floridana L. No symptoms Mild mosaic severe mosaic
N. rustica L. chlorotic spot surrounded by yellowing halla s Chlorotic spots without halla Mild mosaic
N. tabacum cv. Samsun NN. mild mosaic and vein clearing Mild mosaic, vein clearing Necrosis and veinal necrosis
C. annumm.cv. California wonder No symptoms Mild mosaic and deeping veins Severe mosaic
D. metel L. Mild mosaic Blister, vein clearing and crinkling Leaf deformation, chlorosis and severe mosaic
L. esculantum L. cv. Castle rock Veinal necrosis and epinasity Leaf-cup shape, crinkle, leaf narrowing, leaf malformation and small leaflets Severe mosaic, leaf deformation and crinkle
Nicondra physaloides Necrosis and crinkle Leaf curl and crinkle Leaf, veinal necrosis and crinkle
N. glutinosa L Mild mosaic Mild mosaic Leaf deformation and chlorosis
Mode of transmission
Sap transmission + + +
Aphid transmission + + +
Physical properties
TIP (C) 64 66 60
DEP (Dil) 10-5 10-4 10-3
LIV (D) 4 6 4
Microscopic examination of infected strips
Crystalline inclusions + + +
Amorphous inclusions + + +
Susceptibility of potato cvs.
Lady Roseatta Vein clearing Vein clearing Necrosis
Diamont Apical necrosis Yellowing Apical necrosis
Spunta Yellowing Yellowing Apical necrosis
Characters PVY-A PVY-B PVY-C
Serological characterization
DAS-ELISA (Polyclonal antibodies)
D. metel L
Diamont
0.181
0.54
1.075
0.52
0.340
0.60
Double diffusion test +++ ++++ +++
Monoclonal antibodies (Serotype
PVYC,O 1.340 1.456 0.975
PVYN 0.175 0.225 0.722
Molecular characterization
RT-PCR
Number of base pairs
859 bp
872 bp
839 bp
NASH
Color Pale purple Intense purple Purple
Density ++ ++++ +++
Diameter (cm) 0.4 0.45 0.45