Determination of distribution pattern of Candidatus Phytoplasma australasiae in tomato plants as revealed by Real-time PCR
Subject Areas : BacteriologyNazanin Ebadi 1 , Gilda Najafipour 2 , Mohammad Mehdi Faghihi 3 , Kavous Ayazpour 4
1 - Ph.D. student, Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
2 - Assistant Professor, Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
3 - Assistant Professor, Plant Protection Research Department, Fars Agricultural and Natural Resources Research and Education Center, Agricultural Research Education and Extension Organization (AREEO), Shiraz, Iran.
4 - Assistant Professor, Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
Keywords: Tomato, concentration, Root, phytoplasma, Apical leaves,
Abstract :
Background & Objectives: Tomato big bud is a phytoplasma disease which has been recently distributed in different areas of Iran. This study aimed to determine the concentration and distribution pattern of 'Candidatus Phytoplasma australasiae' within tomato plants for its rapid detection, especially during the incubation period of the disease.Materials & Methods: Three tomato plants were graft-inoculated with 'Candidatus Phytoplasma australasiae' in a factorial experiment in a completely randomized design. At the time intervals of 10, 20, 40, and 70 days post-inoculation, samples of apical leaves, leaves above and below the grafting site and lateral roots were taken from the inoculated plants. The concentration of the phytoplasma and its distribution pattern were evaluated using Real-time PCR. Also, the direct and nested-PCR assays were used and compared for the detection of 'Candidatus Phytoplasma australasiae'.Results: The current study found that after entering into the tomato plants, 'Candidatus Phytoplasma australasiae' moved upward into the apical leaves as well as downward into the roots. The phytoplasma concentration in the apical leaves and roots was higher than the leaves of the middle and lower branches of the inoculated plants. The mean disease incubation period was estimated to be about 40 days. The nested-PCR was more sensitive than direct-PCR in detecting the phytoplasma.Conclusion: For the detection of tomato big bud disease associated with 'Candidatus Phytoplasma australasiae', especially during the incubation period of the disease, it is recommended to sample form apical leaves and roots as well as using nested-PCR.
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Rev Microbiol. 2000; 54: 221-255.
2. Del Serrone P, Marzachi C, Bragaloni M, Galeffi P. Phytoplasma infection of tomato in central
Italy. Phytopathol Mediterr. 2011; 40(2): 137-142.
3. Santos-Cervantes ME, Chávez-Medina JA, Méndez-Lozano J, Leyva- López NE. Detection and
molecular characterization of two little leaf phytoplasma strains associated with pepper and
دنیای میکروبها، سال دوازده شماره چهارم زمستان 1389تعیی الگوی گسترش کاندیداتوس فیتوپلاسما استرالیزیا در گیاه گوجه فرنگی با روش Real-time PCRعبادی و همکاران
913
tomato diseases in Guanajuato and Sinaloa, Mexico. Plant Dis J. 2008; 92(7): 1007-1011.
4. Salehi M, Heydarnejad J, Izadpanah K. Molecular characterization and grouping of 35
phytoplasmas from central and southern provinces of Iran. Iran J Plant Pathol. 2005; 41(1):
62-64.
5. Salehi M, Esmailzadeh Hosseini SA. The first report of a 16SrXII-A phytoplasma associated
with tomato big bud disease in Iran. J Plant Pathol. 2016; 98(3): 692.
6. Salehi E, Salehi M, Taghavi SM, Izadpanah K. A 16SrII-D Phytoplasma strain associated with
tomato witches' broom in Bushehr province, Iran. J Crop Prot. 2014; 3(3): 377-388.
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province. Proceeding of the 20th Iran Plant Protection Congress. 4-7 September, Shiraz, Iran.
569.
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associated with Cannabis sativa witches' broom disease in Iran. Plant Pathol J. 2011; 10(4):
161-167.
9. Davoodi A, Panjekeh N, Moslemkhani K, Taheri AH. Detection and molecular characterization
of tomato big bud disease in Qazvin province. J Crop Prot. 2019; 8(4): 379-388.
10. Salehi E, Salehi M, Masoumi M. Biological and molecular characterization of the
phytoplasma associated with tomato big bud disease in Zanjan province, Iran. Iran J Plant
Pathol. 2016; 52 (3): 415-427.
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Razavi and Khorasan-e Shomali provinces. Proceeding of the 19th Iranian Plant Protection
Congress. 31 July-3 August, Tehran, Iran.
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proliferation (16S rVI) and pigeon pea witches' broom (16SrIX) phytoplasma groups with
tomato big bud disease in Iran. Iran J Plant Pathol. 2014; 50 (2): 77-89.
14. Dehghani A, Salehi M. Tomato big bud disease in Lorestan province. Iran J Plant Pathol.
2011; 47(4): 483.
15. Wei W, Kakizawa S, Suzuki S, Jung HY, Nishigawa H, Miyata SI, Oshima K, Ugaki M, Hibi
T, Namba S. In planta dynamic analysis of onion yellows phytoplasma using localized
دنیای میکروبها، سال دوازده شماره چهارم زمستان 1389تعیی الگوی گسترش کاندیداتوس فیتوپلاسما استرالیزیا در گیاه گوجه فرنگی با روش Real-time PCRعبادی و همکاران
933
inoculation by insect transmission. Phytopathol. 2004; 94(3): 244-250.
16. Saracco P, Bosco D, Veratti F, Marzachì C. Quantification over time of chrysanthemum
yellows phytoplasma (16Sr-I) in leaves and roots of the host plant Chrysanthemum carinatum
(Schousboe) following inoculation with its insect vector. Physiol Mol Plant Pathol. 2006; 67
(3-5): 212–219.
17. Marcone C. Movement of phytoplasmas and the development of disease in the plant. In:
Weintraub PG, Jones P, editors. Phytoplasmas: Genomes, plant hosts and vectors. CAB
International, Wallingford; 2009: 114-131.
18. Soltani A, Torabi B. Design and analysis of agricultural experiments with the SAS program.
Mashhad University Press; 2014. [In Persian].
19. Salehi E, Izadpanah K, Taghavi, SM, Hamzezarghani H, Afsharifar A. Interaction of witches'
broom disease of lime phytoplasma and lettuce phyllody phytoplasma in mixed infection of
periwinkle plants. Iran J Plant Pathol. 2018; 54(2): 131-146.
20. Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus. 1990; 12(1): 13-15.
21. Deng S, Hiruki C. Amplification of 16S rRNA genes from culturable and non-culturable
Mollicutes. J Microbiol Methods. 1991; 14(1): 53-61.
22. Schneider B, Seemüller E, Smart CD, Kirkpatrick BC. Phylogenetic classification of plant
pathogenic mycoplasma like organisms or phytoplasmas. In: Razin S, Tully JG, editors.
Molecular and diagnostic procedures in mycoplasmology. Academic Press, San Diego; 1995:
369-380.
23. Gunderson DE, Lee IM. Ultrasensitive detection of phytoplasmas by nested-PCR assays using
two universal primer sets. Phytopathol Mediterr. 1996; 35(3): 144-151.
24. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM =blastn & PAGE_ TYPE= BlastSearch &
LINK_LOC=blasthome
25. Salehi E, Izadpanah K, Taghavi, SM, Hamzezarghani H, Afsharifar A. Interaction of witches'
broom disease of lime phytoplasma and lettuce phyllody phytoplasma in mixed infection of
periwinkle plants. Iran J Plant Pathol. 2018; 54(2): 131-146.
26. Tatineni S, Sagaram US, Gowda S, Robertson CJ, Dawson WO, Iwanami T, Wang N. In
planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain
reaction (PCR) and Real-time PCR. Phytopathology. 2008; 98(5): 592-599.
27. Liefting LW, Andersen MT, Beever RE, Gardner RC, Forster RL. Sequence heterogeneity in
the two 16S rRNA genes of Phormium yellow leaf phytoplasma. Appl Environ Microbiol.
1. Lee IM, Davis RE, Gundersen-Rindal DE. Phytoplasma: phytopathogenic mollicutes. Annu
Rev Microbiol. 2000; 54: 221-255.
2. Del Serrone P, Marzachi C, Bragaloni M, Galeffi P. Phytoplasma infection of tomato in central
Italy. Phytopathol Mediterr. 2011; 40(2): 137-142.
3. Santos-Cervantes ME, Chávez-Medina JA, Méndez-Lozano J, Leyva- López NE. Detection and
molecular characterization of two little leaf phytoplasma strains associated with pepper and
دنیای میکروبها، سال دوازده شماره چهارم زمستان 1389تعیی الگوی گسترش کاندیداتوس فیتوپلاسما استرالیزیا در گیاه گوجه فرنگی با روش Real-time PCRعبادی و همکاران
913
tomato diseases in Guanajuato and Sinaloa, Mexico. Plant Dis J. 2008; 92(7): 1007-1011.
4. Salehi M, Heydarnejad J, Izadpanah K. Molecular characterization and grouping of 35
phytoplasmas from central and southern provinces of Iran. Iran J Plant Pathol. 2005; 41(1):
62-64.
5. Salehi M, Esmailzadeh Hosseini SA. The first report of a 16SrXII-A phytoplasma associated
with tomato big bud disease in Iran. J Plant Pathol. 2016; 98(3): 692.
6. Salehi E, Salehi M, Taghavi SM, Izadpanah K. A 16SrII-D Phytoplasma strain associated with
tomato witches' broom in Bushehr province, Iran. J Crop Prot. 2014; 3(3): 377-388.
7. Akbarpour K, Salehi E, Salehi M. 2012. First report of tomato big bud disease in Hormozgan
province. Proceeding of the 20th Iran Plant Protection Congress. 4-7 September, Shiraz, Iran.
569.
8. Vali Sichani F, Bahar M, Zirak L. Characterization of stolbur (16SrXII) group phytoplasmas
associated with Cannabis sativa witches' broom disease in Iran. Plant Pathol J. 2011; 10(4):
161-167.
9. Davoodi A, Panjekeh N, Moslemkhani K, Taheri AH. Detection and molecular characterization
of tomato big bud disease in Qazvin province. J Crop Prot. 2019; 8(4): 379-388.
10. Salehi E, Salehi M, Masoumi M. Biological and molecular characterization of the
phytoplasma associated with tomato big bud disease in Zanjan province, Iran. Iran J Plant
Pathol. 2016; 52 (3): 415-427.
11. Jamshidi E, Jafarpour B, Roohani H, Salehi, M. 2011. Tomato big bud disease in Khorasan-e
Razavi and Khorasan-e Shomali provinces. Proceeding of the 19th Iranian Plant Protection
Congress. 31 July-3 August, Tehran, Iran.
12. Rashidi M, Ghosta Y, Bahar M. 2006. Russain olive (Elaeagnus angustifolia L.), a new host
for phytoplasma from Iran. Proceeding of the 19th Iranian Plant Protection Congress. 2-5
September, Karaj, Iran. 349.
13. Jamshidi E, Jafarpoor B, Roohani H, Salehi M. Association of members of clover
proliferation (16S rVI) and pigeon pea witches' broom (16SrIX) phytoplasma groups with
tomato big bud disease in Iran. Iran J Plant Pathol. 2014; 50 (2): 77-89.
14. Dehghani A, Salehi M. Tomato big bud disease in Lorestan province. Iran J Plant Pathol.
2011; 47(4): 483.
15. Wei W, Kakizawa S, Suzuki S, Jung HY, Nishigawa H, Miyata SI, Oshima K, Ugaki M, Hibi
T, Namba S. In planta dynamic analysis of onion yellows phytoplasma using localized
دنیای میکروبها، سال دوازده شماره چهارم زمستان 1389تعیی الگوی گسترش کاندیداتوس فیتوپلاسما استرالیزیا در گیاه گوجه فرنگی با روش Real-time PCRعبادی و همکاران
933
inoculation by insect transmission. Phytopathol. 2004; 94(3): 244-250.
16. Saracco P, Bosco D, Veratti F, Marzachì C. Quantification over time of chrysanthemum
yellows phytoplasma (16Sr-I) in leaves and roots of the host plant Chrysanthemum carinatum
(Schousboe) following inoculation with its insect vector. Physiol Mol Plant Pathol. 2006; 67
(3-5): 212–219.
17. Marcone C. Movement of phytoplasmas and the development of disease in the plant. In:
Weintraub PG, Jones P, editors. Phytoplasmas: Genomes, plant hosts and vectors. CAB
International, Wallingford; 2009: 114-131.
18. Soltani A, Torabi B. Design and analysis of agricultural experiments with the SAS program.
Mashhad University Press; 2014. [In Persian].
19. Salehi E, Izadpanah K, Taghavi, SM, Hamzezarghani H, Afsharifar A. Interaction of witches'
broom disease of lime phytoplasma and lettuce phyllody phytoplasma in mixed infection of
periwinkle plants. Iran J Plant Pathol. 2018; 54(2): 131-146.
20. Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus. 1990; 12(1): 13-15.
21. Deng S, Hiruki C. Amplification of 16S rRNA genes from culturable and non-culturable
Mollicutes. J Microbiol Methods. 1991; 14(1): 53-61.
22. Schneider B, Seemüller E, Smart CD, Kirkpatrick BC. Phylogenetic classification of plant
pathogenic mycoplasma like organisms or phytoplasmas. In: Razin S, Tully JG, editors.
Molecular and diagnostic procedures in mycoplasmology. Academic Press, San Diego; 1995:
369-380.
23. Gunderson DE, Lee IM. Ultrasensitive detection of phytoplasmas by nested-PCR assays using
two universal primer sets. Phytopathol Mediterr. 1996; 35(3): 144-151.
24. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM =blastn & PAGE_ TYPE= BlastSearch &
LINK_LOC=blasthome
25. Salehi E, Izadpanah K, Taghavi, SM, Hamzezarghani H, Afsharifar A. Interaction of witches'
broom disease of lime phytoplasma and lettuce phyllody phytoplasma in mixed infection of
periwinkle plants. Iran J Plant Pathol. 2018; 54(2): 131-146.
26. Tatineni S, Sagaram US, Gowda S, Robertson CJ, Dawson WO, Iwanami T, Wang N. In
planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain
reaction (PCR) and Real-time PCR. Phytopathology. 2008; 98(5): 592-599.
27. Liefting LW, Andersen MT, Beever RE, Gardner RC, Forster RL. Sequence heterogeneity in
the two 16S rRNA genes of Phormium yellow leaf phytoplasma. Appl Environ Microbiol.
