تاثیر باکتری های اندوفیت برنج در کنترل زانتوموناس اریزی پاتووار اریزی و الگوی بیان ژن فنیل آلانین آمونیالیاز در شرایط همزیستی با باسیلوس سوبتیلیس
محورهای موضوعی : میکروب شناسی مولکولیحدیث یوسفی 1 , نادر حسن زاده 2 , کیوان بهبودی 3 , فرید بیکی فیروزجاهی 4
1 - دانشجوی دکتری، گروه گیاهپزشکی، دانشکده علوم کشاورزی و صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران.
2 - دانشیار، گروه گیاهپزشکی، دانشکده علوم کشاورزی و صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران.
3 - دانشیار، گروه گیاهپزشکی، دانشکده علوم و مهندسی کشاورزی، پردیس کشاورزی و منابع طبیعی، دانشگاه تهران، تهران، ایران.
4 - استادیار، موسسه تحقیقات گیاهپزشکی کشور، سازمان تحقیقات آموزش و ترویج کشاورزی، تهران، ایران.
کلید واژه: باسیلوس سوبتیلیس, مقاومت القایی, بهبود رشد گیاه, باکتری های اندوفیت برنج,
چکیده مقاله :
سابقه و هدف: بیماری بلایت باکتریایی برنج یکی از عوامل جدی محدود کننده تولید جهانی برنج است. موثرترین روش کنترل این بیماری استفاده از ارقام مقاوم هست. اما به دلیل تغییر پذیری زیاد و تکامل سریع نژادهای بیماریزا، این ارقام در شرایط مزرعه پایدار نیستند. این مطالعه با هدف ارزیابی توانایی باکتری های اندوفیت برنج در بهبود رشد گیاه و کنترل زیستی باکتری زانتوموناس اریزی پاتووار اریزی انجام گردید. مواد و روش‌ها: ابتدا غربالگری جدایه های اندوفیت برای فعالیت آنتاگونیستی روی باکتری زانتوموناس در محیط کشت تریپتیک سویا آگار انجام شد. سپس تحریک رشد گیاه توسط اندوفیت های باکتریایی تحت شرایط اتاقک رشد و گلخانه تعیین گردید. همچنین اثر جدایه ها بر شاخص شدت بیماری و برخی عوامل رشد برنج در شرایط گلخانه مشخص گردید. در نهایت میزان بیان ژن فنیل آلانین آمونیالیاز توسط جدایه اندوفیت باسیلوس سوبتیلیس با روش real-time PCR مورد ارزیابی قرار گرفت. یافته‌ها: از نظر شاخص قدرت گیاهچه بذرهای برنج، جدایه های OS40، OS23، OS43 و OS31 اثرات آماری معنی داری نسبت به شاهد داشتند. همچنین کاربرد اندوفیت های OS3، OS23، OS31 و OS40 موجب افزایش شاخص های رشد گردید. تلقیح جدایه های باکتریایی OS40 و OS23 در کاهش شدت بیماری و تحریک رشد گیاه موفق تر عمل کردند. سطح بیان ژن PAL در دوره آزمون در گیاهان تیمار OS40 به طور معنی داری بیشتر و سریع تر از گیاهان تیمار زانتوموناس به تنهایی بود. نتیجه گیری: باکتری های اندوفیت برنج توانستند رشد گیاهان را افزایش و بیماری بلایت را کاهش دهند پس می توانند به عنوان راهکاری امید بخش و سازگار با محیط زیست در توسعه کشاورزی پایدار در نظر گرفته شوند.
Background & Objectives: Rice bacterial blight is one of the major factors limiting global rice production. Though the most effective method for controlling this disease is using resistant varieties, they are not stable in farm conditions due to high variability and rapid evolution of pathogenic races. This study was aimed to evaluate rice endophyte bacteria's ability to improving plant growth and biologically controlling Xanthomonas oryzae pv. oryzae.Materials & Methods: At the first screening of endophyte isolates was performed to evaluate antagonistic activity against Xanthomonas in tryptic soy agar medium. The stimulation of plant growth by bacterial endophytes was assessed under the growth chamber and greenhouse conditions. The effect of bacterial isolates on disease severity and some rice growth factors under greenhouse conditions were determined, as well. In the final stage, the expression of the Phenylalanine ammonia-lyase gene by endophytic isolate Bacillus subtilis was evaluated using real-time PCR.Results: In terms of rice seed seedling vigor index, OS40, OS23, OS43, and OS31 isolates had a significant statistical effect as compared to control. Moreover, the use of OS3, OS23, OS31, and OS40 endophytes increased plant growth parameters. OS40 and OS23 bacterial inoculants were more successful in reducing the severity of the disease and the promotion of plant growth. PAL expression level during the experiment period was significantly higher and faster in plants treated with OS40than those treated only with Xanthomonas- isolates.Conclusion: Rice bacterial endophytes could increase plant growth and reduce disease severity, thus they can be considered as a promising and environmentally- a friendly strategy for sustainable agriculture development.
of blast disease resistance in rice plants by endophyte fungus Piriformospora indica. Iran J
Plant Path. 2014; 50(3): 127-129.
2. Ou SH. Rice Diseases. 2nd ed. Kew- Surrey- England. Common Wealth Mycological Institute;
1985.
3. Peng H, Chen ZH, Fang ZH, Zhou J, Xi ZH, Gao L, Chen L, Li L, Li T, Zhai W, Zhang W.
Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection.
Sci Rep. 2015; 5: 12165.
4. Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kleopper W. Bacterial endophytes in
agricultural crops. Can J Microbiol. 1997; 43: 895-914.
5. Schulz B, Boyle C. What are endophytes? In: Schulz BJE, Boyle CJC, Sieber TN. (eds).
Microbial Root Endophytes. Berlin Germany. Springer; 2006.
6. Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim YO,
Redman RS. Stress tolerance in plants via habitat-adapted symbiosis. ISME J. 2008; 2(4):
404-416.
7. Rodriguez RJ, White JFJr, Arnold AE, Redman RS. Fungal endophytes: diversity and functional
roles. New Phytol. 2009; 182(2): 314-330.
8. Sturz AV, Christie BR, Matheson BG, Nowak J. Biodiversity of endophytic bacteria which
colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol
Fertil Soils. 1997; 25: 13-19.
9. Rosenblueth M, Martínez-Romero E. Bacterial endophytes and their interactions with hosts.
MPMI. 2006; 19(8): 827-837.
10. Reinhold-Hurek B, Hurek T. Interactions of gramineous plants with Azoarcus spp. and other
diazotrophs: Identification, localization, and perspectives to study their function. Crit Rev Plant
Sci. 1998; 17: 29-54.
11. Sturz AV, Matheson BG. Populations of endophytic bacteria which influence host-resistance to
Erwinia-induced bacterial soft rot in potato tubers. Plant Soil. 1996; 184: 265-271.
12. Duijff B J, Gianinazzi-Pearson V, Lemanceau P. Involvement formed pea roots after challenge
with Fusarium oxysporum f. sp. pisi and Pythium ultimum. Phytopathol. 1997; 86: 114-178.
13. Krishnamurthy K, Gnanamanickam SS. Biological control of sheath blight of rice: induction of
systemic resistance in rice by plant-associated Pseudomonas spp. Curr Sci.1997; 72: 331-334.
14. Azevedo JL, Maccheroni J Jr, Pereira O, Ara WL. Endophytic microorganisms: a review on
insect control and recent advances on tropical plants. Electr J Biotech. 2000; 3: 40-65.
15. Hallmann J, Quadt-Hallmann A, Rodr´ıguez-K´abana R, Kloepper JW. Interactions between
Meloidogyne incognita and endophytic bacteria in cotton and cucumber. Soil Biol
Biochem. 1998; 30: 925-937.
16. Chanway C.P. Inoculation of tree roots with plant growth promoting soil bacteria: an emerging
technology for reforestation. Forest Sci. 1997; 43: 99-112.
17. Wei G, Kloepper JW, Tuzun S. Induced systemic resistance to cucumber diseases and
increased plant growth by plant growth-promoting rhizobacteria under field conditions.
Phytopathol. 1996; 86: 221-224.
18. Zehnder GW, Murphy JF, Sikora EJ, Kloepper JW. Application of rhizobacteria for induced
resistance. Eur J Plant Pathol. 2001; 107: 39-50.
19. Murphy JF, Reddy M, Ryu CM, Kloepper JW, Li R. Rhizobacteria-mediated growth
promotion of tomato leads to protection against Cucumber mosaic virus. Phytopathol. 2003; 93:
1301-1307.
20. Firdous J, Bhore Subhash J. Screening of cultivable endophytic bacterial isolates for their plant
growth promoting activity in rice. Indian J Agric Res. 2017; 51(5): 413-418.
21. McInroy JA, Kloepper JW. Population dynamics of endophytic bacteria in field- grown sweet
corn and cotton. Can J Microbiol. 1995; 41: 895-901.
22. ISTA. Proceedings of the international Seed Testing Association, International Rules for Seed
Testing. Seed Science and Technology. 1993; 21: 25-30.
23. Baki AAA, Anderson JD. Vigour determination in soybean seed by multiple criteria. Crop Sci.
1973; 31: 630–633.
24. Chithrashree-Udayashankar AC, Chandra-Nayaka S, Reddy MS, Srinivas C. Plant
growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial
leaf blight caused by Xanthomonas oryzae pv. oryzae. Biol Control. 2011; 59: 114-122.
25. Chung EJ, Hossain MT, Khan A, Kim KH, Jeon CO, Chung YR. Bacillus oryzicola sp. nov.,
an endophytic bacterium isolated from the root of rice with anti-microbial, plant-growth
promoting and systemic resistance-inducing activities in rice. Plant Pathol J. 2015; 31:
152-164.
26. Kauffman HE, Reddy APK, Hsieh SPY, Merca SD. An improved technique for evaluating
resistance of rice varieties to Xanthomonas oryzae. Plant Dis Reporter. 1973; 57: 537-541.
27. Fang ZD, Xu ZG, Guo CJ, Yin SZ, Wu SZ, Xu XM, Zhang Q. Studies on pathotypes of
Xanthomonas campestris pv. oryzae in China. Acta Phytopathol Sin. 1990; 20: 81-88.
28. Elboutahiri N, Thami-Alami I, Zaïd E, M.Udupa S. Genotypic characterization of indigenous
Sinorhizobium meliloti and Rhizobium sullae by rep-PCR, RAPD and ARDRA analyses. Afr J
Biotechnol. 2009; 8(6): 979-985.
29. Palacio-Bielsa A, Cambra MA, López MM. PCR detection and identification of plant
pathogenic bacteria: updated review of protocols (1989-2007). J Plant Pathol. 2009; 91(2):
249-297.
30. Hossain MT, Khan A, Chung EJ, Harun-Or Rashid Md, Chung YR. Biological control of rice
bakanae by an endophytic Bacillus oryzicola YC7007. Plant Pathol J. 2016; 32(3): 228-241.
31. Alizadeh H, Salari KH. Induced resistance by β-amino butyric acid (BABA) against Fusarium
stem and root of Cucumber. IJPPS. 2014; 45(2): 299-307.
32. SongA, Xue G, Cui P, Fan F, Liu H, YinCH, Sun W, Liang Y. The role of silicon in
enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice. Sci Rep. 2016; 6:
24640.
33. Yuan J, Reed A, Chen F, Stewart Jr CN. Statistical analysis of real time PCR data. MBC
Bioanformatic. 2006; 7: 85.
34. Barka EA, Gognies S, Nowak J, Audran JC, Belarbi A. Inhibitory effect of endophytic
bacteria on Botrytis cinerea and its influence to promote the grape vine growth. Biol Control.
2002; 24: 135-142.
35. Oliveira ALM, Canuto EL, Reis VM, Baldani JI. Response of micropropagated sugarcane
varieties to inoculation with endophytic diazotrophic bacteria. Braz J Microbiol. 2003; 34:
59-61.
36. Kang SH, Cho HS, Cheong H, Ryu CM, Kim JF, Park SH. Two bacterial endophytes eliciting
both plant growth promotion and plant defense on Pepper (Capsicum annuum L.). J Microbiol
Biotechnol. 2007; 17(1): 96-103.
37. Reiter B, Pfeifer U, Schwab H, Sessitsch A. Response of endophytic bacterial communities in
potato plants to infection with Erwinia carotovora sub sp. atroseptica. Appl Environ
دنیای میکروبها، سال دوازدهم شماره سوم پاییز .1389تاثیر باکتریهای اندوفیت برنج در کنترل زانتوموناس اریزی پاتووار اریزی و الگوی بیان ژن فنیل آلانین آمونیالیاز ....حدیث یوسفی و همکاران
292
Microbiol. 2002; 68: 2261-2268.
38. Compant S, Duf yB, Nowak J, Cle´ment CH, Ait Bark E. Use of plant growth-promoting
bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future
prospects. Appl Environ Microbiol. 2005; 71(9): 4951-4959.
39. Ji SH, Gururani MA, Chun SCh. Isolation and characterization of plant growth promoting
endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res. 2014; 169: 83-98.
40. Melnick RL, Zidack NK, Bailey BA, Maximova SN, Guiltinan M, Backman PA. Bacterial
endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod
rot of cacao. Biol Control. 2008; 46: 46-56.
41. Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MSS, Wang LJ. The phenylpropanoid pathway
and plant defence- A genomics perspective. Mol. Plant Pathol. 2002; 3: 371-390.
42. Van Loon LC, Bakker PAH M, Pieterse CMJ. Systemic resistance induced by rhizosphere
bacteria. Ann Rev Phytopathol. 1998; 36: 453-483.
43. Gond SK, Bergen MS, Torres MS, White Jr JF. Endophytic Bacillus spp. produce antifungal
lipopeptides and induce host defence gene expression in maize. Microbiol Res. 2015; 172:
79-87.
_||_
of blast disease resistance in rice plants by endophyte fungus Piriformospora indica. Iran J
Plant Path. 2014; 50(3): 127-129.
2. Ou SH. Rice Diseases. 2nd ed. Kew- Surrey- England. Common Wealth Mycological Institute;
1985.
3. Peng H, Chen ZH, Fang ZH, Zhou J, Xi ZH, Gao L, Chen L, Li L, Li T, Zhai W, Zhang W.
Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection.
Sci Rep. 2015; 5: 12165.
4. Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kleopper W. Bacterial endophytes in
agricultural crops. Can J Microbiol. 1997; 43: 895-914.
5. Schulz B, Boyle C. What are endophytes? In: Schulz BJE, Boyle CJC, Sieber TN. (eds).
Microbial Root Endophytes. Berlin Germany. Springer; 2006.
6. Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim YO,
Redman RS. Stress tolerance in plants via habitat-adapted symbiosis. ISME J. 2008; 2(4):
404-416.
7. Rodriguez RJ, White JFJr, Arnold AE, Redman RS. Fungal endophytes: diversity and functional
roles. New Phytol. 2009; 182(2): 314-330.
8. Sturz AV, Christie BR, Matheson BG, Nowak J. Biodiversity of endophytic bacteria which
colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol
Fertil Soils. 1997; 25: 13-19.
9. Rosenblueth M, Martínez-Romero E. Bacterial endophytes and their interactions with hosts.
MPMI. 2006; 19(8): 827-837.
10. Reinhold-Hurek B, Hurek T. Interactions of gramineous plants with Azoarcus spp. and other
diazotrophs: Identification, localization, and perspectives to study their function. Crit Rev Plant
Sci. 1998; 17: 29-54.
11. Sturz AV, Matheson BG. Populations of endophytic bacteria which influence host-resistance to
Erwinia-induced bacterial soft rot in potato tubers. Plant Soil. 1996; 184: 265-271.
12. Duijff B J, Gianinazzi-Pearson V, Lemanceau P. Involvement formed pea roots after challenge
with Fusarium oxysporum f. sp. pisi and Pythium ultimum. Phytopathol. 1997; 86: 114-178.
13. Krishnamurthy K, Gnanamanickam SS. Biological control of sheath blight of rice: induction of
systemic resistance in rice by plant-associated Pseudomonas spp. Curr Sci.1997; 72: 331-334.
14. Azevedo JL, Maccheroni J Jr, Pereira O, Ara WL. Endophytic microorganisms: a review on
insect control and recent advances on tropical plants. Electr J Biotech. 2000; 3: 40-65.
15. Hallmann J, Quadt-Hallmann A, Rodr´ıguez-K´abana R, Kloepper JW. Interactions between
Meloidogyne incognita and endophytic bacteria in cotton and cucumber. Soil Biol
Biochem. 1998; 30: 925-937.
16. Chanway C.P. Inoculation of tree roots with plant growth promoting soil bacteria: an emerging
technology for reforestation. Forest Sci. 1997; 43: 99-112.
17. Wei G, Kloepper JW, Tuzun S. Induced systemic resistance to cucumber diseases and
increased plant growth by plant growth-promoting rhizobacteria under field conditions.
Phytopathol. 1996; 86: 221-224.
18. Zehnder GW, Murphy JF, Sikora EJ, Kloepper JW. Application of rhizobacteria for induced
resistance. Eur J Plant Pathol. 2001; 107: 39-50.
19. Murphy JF, Reddy M, Ryu CM, Kloepper JW, Li R. Rhizobacteria-mediated growth
promotion of tomato leads to protection against Cucumber mosaic virus. Phytopathol. 2003; 93:
1301-1307.
20. Firdous J, Bhore Subhash J. Screening of cultivable endophytic bacterial isolates for their plant
growth promoting activity in rice. Indian J Agric Res. 2017; 51(5): 413-418.
21. McInroy JA, Kloepper JW. Population dynamics of endophytic bacteria in field- grown sweet
corn and cotton. Can J Microbiol. 1995; 41: 895-901.
22. ISTA. Proceedings of the international Seed Testing Association, International Rules for Seed
Testing. Seed Science and Technology. 1993; 21: 25-30.
23. Baki AAA, Anderson JD. Vigour determination in soybean seed by multiple criteria. Crop Sci.
1973; 31: 630–633.
24. Chithrashree-Udayashankar AC, Chandra-Nayaka S, Reddy MS, Srinivas C. Plant
growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial
leaf blight caused by Xanthomonas oryzae pv. oryzae. Biol Control. 2011; 59: 114-122.
25. Chung EJ, Hossain MT, Khan A, Kim KH, Jeon CO, Chung YR. Bacillus oryzicola sp. nov.,
an endophytic bacterium isolated from the root of rice with anti-microbial, plant-growth
promoting and systemic resistance-inducing activities in rice. Plant Pathol J. 2015; 31:
152-164.
26. Kauffman HE, Reddy APK, Hsieh SPY, Merca SD. An improved technique for evaluating
resistance of rice varieties to Xanthomonas oryzae. Plant Dis Reporter. 1973; 57: 537-541.
27. Fang ZD, Xu ZG, Guo CJ, Yin SZ, Wu SZ, Xu XM, Zhang Q. Studies on pathotypes of
Xanthomonas campestris pv. oryzae in China. Acta Phytopathol Sin. 1990; 20: 81-88.
28. Elboutahiri N, Thami-Alami I, Zaïd E, M.Udupa S. Genotypic characterization of indigenous
Sinorhizobium meliloti and Rhizobium sullae by rep-PCR, RAPD and ARDRA analyses. Afr J
Biotechnol. 2009; 8(6): 979-985.
29. Palacio-Bielsa A, Cambra MA, López MM. PCR detection and identification of plant
pathogenic bacteria: updated review of protocols (1989-2007). J Plant Pathol. 2009; 91(2):
249-297.
30. Hossain MT, Khan A, Chung EJ, Harun-Or Rashid Md, Chung YR. Biological control of rice
bakanae by an endophytic Bacillus oryzicola YC7007. Plant Pathol J. 2016; 32(3): 228-241.
31. Alizadeh H, Salari KH. Induced resistance by β-amino butyric acid (BABA) against Fusarium
stem and root of Cucumber. IJPPS. 2014; 45(2): 299-307.
32. SongA, Xue G, Cui P, Fan F, Liu H, YinCH, Sun W, Liang Y. The role of silicon in
enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice. Sci Rep. 2016; 6:
24640.
33. Yuan J, Reed A, Chen F, Stewart Jr CN. Statistical analysis of real time PCR data. MBC
Bioanformatic. 2006; 7: 85.
34. Barka EA, Gognies S, Nowak J, Audran JC, Belarbi A. Inhibitory effect of endophytic
bacteria on Botrytis cinerea and its influence to promote the grape vine growth. Biol Control.
2002; 24: 135-142.
35. Oliveira ALM, Canuto EL, Reis VM, Baldani JI. Response of micropropagated sugarcane
varieties to inoculation with endophytic diazotrophic bacteria. Braz J Microbiol. 2003; 34:
59-61.
36. Kang SH, Cho HS, Cheong H, Ryu CM, Kim JF, Park SH. Two bacterial endophytes eliciting
both plant growth promotion and plant defense on Pepper (Capsicum annuum L.). J Microbiol
Biotechnol. 2007; 17(1): 96-103.
37. Reiter B, Pfeifer U, Schwab H, Sessitsch A. Response of endophytic bacterial communities in
potato plants to infection with Erwinia carotovora sub sp. atroseptica. Appl Environ
دنیای میکروبها، سال دوازدهم شماره سوم پاییز .1389تاثیر باکتریهای اندوفیت برنج در کنترل زانتوموناس اریزی پاتووار اریزی و الگوی بیان ژن فنیل آلانین آمونیالیاز ....حدیث یوسفی و همکاران
292
Microbiol. 2002; 68: 2261-2268.
38. Compant S, Duf yB, Nowak J, Cle´ment CH, Ait Bark E. Use of plant growth-promoting
bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future
prospects. Appl Environ Microbiol. 2005; 71(9): 4951-4959.
39. Ji SH, Gururani MA, Chun SCh. Isolation and characterization of plant growth promoting
endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res. 2014; 169: 83-98.
40. Melnick RL, Zidack NK, Bailey BA, Maximova SN, Guiltinan M, Backman PA. Bacterial
endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod
rot of cacao. Biol Control. 2008; 46: 46-56.
41. Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MSS, Wang LJ. The phenylpropanoid pathway
and plant defence- A genomics perspective. Mol. Plant Pathol. 2002; 3: 371-390.
42. Van Loon LC, Bakker PAH M, Pieterse CMJ. Systemic resistance induced by rhizosphere
bacteria. Ann Rev Phytopathol. 1998; 36: 453-483.
43. Gond SK, Bergen MS, Torres MS, White Jr JF. Endophytic Bacillus spp. produce antifungal
lipopeptides and induce host defence gene expression in maize. Microbiol Res. 2015; 172:
79-87.