مطالعه‌ی تاثیر برخی متابولیت‌های تولید شده توسط Pseudomonas tolaasii و باکتری‌های همراه روی قارچ خوراکی (Agaricus bisporus)

محورهای موضوعی : دو فصلنامه تحقیقات بیماریهای گیاهی

شادی تجلی پور ¹ (دانش آموخته کارشناسی ارشد، گروه بیماری شناسی گیاهی، دانشگاه آزاد اسلامی، دانشکده کشاورزی و منابع طبیعی، واحد علوم و تحقیقات تهران، تهران، ایران.)
نادر حسن زاده ² (دانشیار، گروه بیماری شناسی گیاهی، دانشکده کشاورزی و منابع طبیعی، واحد علوم و تحقیقات تهران، دانشگاه آزاد اسلامی، تهران، ایران.)
اصغر حیدری ³ (دانشیار، گروه بیماری شناسی گیاهی، موسسه تحقیقات گیاهپزشکی کشور، تهران، ایران.)
حسین خباز جلفایی ⁴ (مربی، گروه بیماری شناسی گیاهی، موسسه تحقیقات گیاهپزشکی کشور، تهران، ایران.)

تاریخ دریافت : 1394/11/12 تاریخ پذیرش : 1394/11/12 تاریخ انتشار : 1395/03/01

کلید واژه: قارچ دکمه­ای سفید, لکه قهوه­ای, Agaricus bisporus, Pseudomonas reactans, Pseudomonas tolaasii,

چکیده مقاله :

دراین تحقیق به بررسی برخی متابولیت­های Pseudomonas tolaasiiعامل لکه قهوه­ای قارچ خوراکی دکمه­ای و نیز Pseudomonas reactansباکتری همراه قارچ خوراکی دکمه­ای، پرداخته شد. خصوصیات شاخصP. Tolaasiiشامل ایجاد لکه قهوه­ای و نقاط فرو رفته روی کلاهک قارچ خوراکی دکمه­ای A. bisporus، تولید تولاسین، همولیزاریتروسیت­ها، تولید رسوب سفید رنگ در برابر باکتری P. reactans است. در مطالعه­ی حاضر تأثیرP. Tolaasiiو متابولیت حاصل از آن بر میزان رشد میسلیوم Agaricus bisporus مورد بررسی قرار گرفت. پس از استخراج ترکیبات ناخالص متابولیتی یاWhite Line Inducing Principle (WLIP)، تولید شده توسط P. reactansو نیز توکسین تولاسین، آزمون­های متعددی جهت بررسی تأثیر متقابل و غیرمستقیم این ترکیبات انجام گرفت. همچنین ژن rpoBمتعلق به جدایه بیماریزای Pseudomonas sp. p2 که از قارچ مذکور جداسازی شده بود با استفاده از جفت پرایمر LAPS27 و LAPS تکثیر، تعیین توالی و در بانک ژن NCBI ثبت گردید. تأثیر ترکیب WLIP ناخالص حاصل از P. reactans Pr 5 و جدایه استاندارد P. reactans NCPPB1311 بر بلوک­های A. bisporus وسیب­زمینی بطور جداگانه مورد بررسی قرار گرفت. در زیست سنجی­های مختلف مشاهده شد بلوک­های قارچ مایه­زنی شده با توکسین بیماریزاترین استرین بومی P. tolaasii Pt 6 تولید علایم لکه قهوه­ای تیره و نقاط کمی فرو رفته نمودند. برعکس، در برابرترکیب WLIP استرین P. reactans Pr 5 تغییر رنگ جزئی و خفیف بود. از یافته­های مهم دیگر، بکارگیری همزمان تیمار P. reactans A6 و ترکیب تولاسین هر دو جدایۀ بومی و استاندارد بر بلوک قارچ بود که منجر به توقف علایم آشکار قهوه­ای شدن ناشی از تولاسین شد و با خاصیت بازدارندگی خود، موجب مهار فعالیت تولاسین گردید. این نتایج نشان­دهنده اهمیت باکتری­های همراه در بازداری رشد و توقف بیماریزائی باکتری P. tolaasiiاست
 
.

چکیده انگلیسی:

In this research some metabolites of Pseudomonas tolaasii the causal agent of mushroom bacterial blotch disease and Pseudomonas reactans associated with it were investigated. Characteristic features of P. tolaasii included brown pit formation on button mushroom caps, tolaasin production, lysis of erythrocytes, and white line induction in agar plate in presence of Pseudomonas reactans. Further, the metabolites of P. tolaasii and mushroom-associated bacteria were extracted and their effects were evaluated against the blocks of the mushroom A. bisporus. It was noticed that the strains of P. reactans also produced a lipodepsipeptide, WLIP. Certain complementary tests were also conducted to confirm the presence of these compounds in products extracted. The rpoB gene of a pathogenic strain of Pseudomonas sp. p2 isolated from the same mushroom was amplified using the primer pair of LAPS and LAPS27 and sequenced and deposited in Genebank at NCBI. Effect of impure WLIP of the strain P. reactans Pr 5 and standard strain of P. reactans NCPPB1311 were examined on blocks of A. bisporus and potato tubers. In bioassays, the tolaasin of strain P. tolaasii Pt 6 created both brown blotch and slight pitting symptoms. Whereas, WLIP of the P. reactans Pr 5 could only induce slight discoloration. Another important finding was that simultaneous application of P. reactans A6 in combination with tolaasins of P. tolaasii Pt 6 or P. tolaasii NCPPB2192 resulted in suppression of brown blotch symptoms on treated blocks with P. tolaasii. Results indicate the importance of associated bacteria in suppression of disease by P. tolaasii.

منابع و مأخذ:

1. Andolfi A, Alessio Cimmino A, Lo Cantore P, Iacobellis NS and Evidente A. 2008. Bioactive and structural metabolites of Pseudomonas and Burkholderia species causal agents of cultivated mushrooms diseases. Perspectives Medicinal Chemistry 2: 81–112.


2. Bassarello G, Lazzaroni S, Bifulco G, Lo Cantore P, Iacobellis NS, Riccio R, Gomes-Paloma L and Evidente A. 2004. Tolaasins A-E, five new lipodepsipeptides produced by Pseudomonas tolaasii. American Chemical Society and American Society of Pharmacognosy 67: 811–816.


3. Beelman RB, Guthrie BD and Royse DJ. 1989. Influence of bacterial populations on postharvest deterioration of fresh mushrooms. Mushroom Science 12: 655–665.


4. Brodey CL, Rainey PB, Tester M and Johnstone K. 1991. Bacterial blotch disease of the cultivated mushroom is caused by an ion channel forming lipodepsipeptide toxin. Molecular Plant-Microbe Interactions 4: 407–411.


5. Ercolani GL. 1970. Primi risultati di osservazioni sulla maculatura batterica dei funghi coltivati (Agaricus bisporus (Lange) Imbach) in Italia: di Pseudomonas tolaasii Paine. Phytopathology Mediterranea 9: 59–61.


6. Fermor TR and Lynch JM. 1988. Bacterial blotch disease of the cultivated mushroom Agaricus bisporus: screening, isolation and characterization of bacterial antagonistic to the pathogen (Pseudomonas tolaasii). Journal of Applied Bacteriology 65: 179–187.


7. Geels FP. 1995. Pseudomonas tolaasii control by kaisugamycin in cultivated mushrooms (Agaricus bisporus). Journal of Applied Bacteriology 79: 38–42.


8. Godfrey SAC, Marshall JW and Klena JD. 2001. Genetic characterization of Pseudomonas "NZ17" – a novel pathogen that results in a brown blotch disease of Agaricus bisporus. Journal of Applied Microbiology 91: 412–420.


9. Grewal PS and Hand P. 1992. Effects of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on the mycelial growth of Agaricus bisporus. Jornal of Applied Bacteriology 72: 173–179.


10. Grewal SIS, Han B and Johnstone K. 1995. Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus. Journal of Bacteriology 177: 4658–4668.


11. Han B, Pain A, Smith J, Grewal S and Johnstone K. 1995. Regulation of phenotypic variation and toxin synthesis in Pseudomonas tolaasii. Indian Journal of Mycology and Plant Pathology 25: 105–106.


12. Hutchison ML and Johnstone K. 1993. Evidence for the involvement of the surface active properties of the extracellular toxin tolaasin in the manifestation of brown blotch disease symptoms by Pseudomonas tolaasii on Agaricus bisporus. Physiological and Molecular Plant Pathology 42: 373–384.


13. Khabbaz-Jolfaee H and Rahimian H. 2002. Brown blotch disease of cultivated mushroom (Agaricus bisporus) in Iran. Iranian Journal of Plant Pathology 38: 1–2 (In Persian).


14. Khabbaz-Jolfaee H, Mohammadi Goltapeh H and Rahimian HA. 2005. Isolation, screening and evaluation of the efficacy of potentially antagonistic bacteria for the biocontrol of brown blotch disease of the cultivated mushroom Agaricus bisporus. Iranian Journal of Plant Pathology 41: 543–559.


15. Kwon S-W, Kim S-H and Go S-J. 2000. PCR assays for detection of Pseudomonas tolaasii and Pseudomonas agarici. Microbiology 28: 89–92.


16. Lo Cantore P, Lazzaroni S, Coraiola M, Dalla Serra, M, Cafarchia C, Evidente A and Lacobellis NS. 2006. Biological characterization of white line inducing principle (WLIP) produced by Pseudomonas reactans NCPPB1311. Molecular Plant-Microbe Interaction 19: 1113–1120.


17. Munsch P and Alatossava T. 2002. Several Pseudomonads, associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic. Microbiology Research 157: 311–315.


18. Nair NG and Fahy PC. 1972. Bacteria antagonistic to Pseudomonas tolaasii and their control of brown blotch of the cultivated mushroom Agaricus bisporus. Journal of Applied Bacteriology 35: 439–442.


19. Nair NG and Fahy PC. 1973. Toxin production by Pseudomonas tolaasii Paine. Australian Journal of Biological Sciences 26: 509–512.               


20. Nair NG. 2004. First report of brown discoloration of Agaricus bisporus caused by Pseudomonas agarici in southern Italy. Phytopathologia Mediterranea 43: 35–38.


21. Obradovic A, Gasic K and Ivanovic M. 2008. Bacterial diseases of Agaricus bisporus in Serbia. Biomedical and Life Sciences. pp. 427–430, In M Fatmi, A Collmer, NS Iacobellis, J Mansfield, J Murillo, NW Schaad, M Ullrich (eds). Pseudomonas syringae Pathovars and Related Pathogens - Identification, Epidemiology and Genomics. Wallingford: Springer Science + Business Media.


22. Paine SG. 1919. Studies in bacteriosis. II: A brown blotch disease of cultivated mushrooms. Annals of Biology 5: 206–219.


23. Rademaker JLW and de Bruijn FJ. 1997. Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis. In: Caetano-Anollés G, Gresshoff PM (eds.): DNA markers: Protocols, Applications and Overviews, J. Wiley & Sons, USA. Chapter 10 p. 151-171.


24. Rahimiyan C, Zarei A and Okhovatian C. 1995. Mushroom brown blotch in Mazandaran. Paper presented at: 12^th Iranian Plant Protection Congress; 2–7 September; Tehran, Iran.


25. Rainey PB, Brodey CL and Johnstone K. 1991. Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii. Physiological and Molecular Plant Pathology 39: 57–70.


26. Schaad NW, Jones JB and Chun W. 2001. Laboratory guide for identification of plant pathogenic bacteria. 3^rd ed. St. Paul: American Phytopathological Society Press, 398 p.


27. Shirata A, Sugaya K, Takasugi M and Monde K. 1995. Isolation and biological activity of toxins produced by a Japanese strain of Pseudomonas tolaasii, the pathogen of bacterial rot of cultivated oyster mushroom. Annals of the Phytopathological Society of Japan 61: 493–502.


28. Soler-Rivas C, Jolivet S, Arpin N, Oliver JM and Wichers HJ. 1999. Biochemical and physiological aspects of brown blotch disease of Agaricus bisporus. FEMS Microbiology Review 23: 591–614.


29. Soler-Rivas C. 1998. Molecular aspects of the bacterial blotch disease of Agaricus bisporus [PhD]. [Lyon (France)]: University Claude-Bernard Lyon 1.


30. Tajalipour Sh, Hassanzadeh N, Heydari A and Khabbaz-Jolfaee H. 2012. Phenotypic and genotypic characterization of Pseudomonas tolaasii, the causal agent of brown blotch disease on button mushroom (Agaricus bisporus). Paper presented at: 19^th Iranian Plant ProtectionCongress; 31 July–3 August; Tehran, Iran.


31. Tajalipour Sh, Hassanzadeh N, Heydari A and Khabbaz-Jolfaee H. 2015. Study on genetic diversity of Pseudomonas tolaasii and Pseudomonas reactans bacteria associated with mushroom brown blotch disease employing ERIC and BOX-PCR techniques. International Journal of Agriculture and Crop Sciences 8: 398–405.


32. Tajalipour Sh, Hassanzadeh N, Khabbaz-Jolfaee H, Heydari A and Ghasemi A. 2014. Biological control of mushroom brown blotch disease using antagonistic bacteria Biocontrol Science and Technology 24: 473–484.


33. Tamura K, Dudley J, Nei M and Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596–1599.


34. Tsukamoto T, Murata H and Shirata A. 2002. Identification of Non-Pseudomonad bacteria from fruit bodies of wild Agaricales Fungi that detoxify tolaasin produced by Pseudomonas tolaasii. Bioscience Biotechnology and Biochemistry 66: 2201–2208.


35. Wells JM, Sapers GM, Fett WF, Butterfield JE, Jones JB, Bouzar H and Miller FC. 1996. Postharvest discoloration of the cultivated mushroom Agaricus bisporus caused by Pseudomonas tolaasii, P. reactans and P. gingeri. Phytopathology 86: 1098–1104.


36. Wong WC and Preece TF. 1979. Identification of Pseudomonas tolaasii: the white line in agar and mushroom tissue block rapid pitting tests. Journal of Applied Bacteriology 47: 401–407.


37. Wong WC and Preece TF. 1982. Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: numbers of the bacterium and symptom development on mushrooms grown in various environments after artificial inoculation. Journal of Applied Bacteriology 53: 87–96.