ارزﯾﺎﺑﯽ ﺗﻮان ﺑﯿﻮﮐﻨﺘﺮل و ﮐﻠﻨﯿﺰاﺳﯿﻮن ﺟﺪاﯾﻪﻫﺎي ﺗﺮﯾﮑﻮدرﻣﺎ ﺑﻬﯿﻨﻪﺳﺎزي ﻣﻮﻟﮑﻮﻟﯽ ﺷﺪه در ﺑﺮاﺑﺮ ﺑﯿﻤﺎري راﯾﺰوﮐﺘﻮﻧﯿﺎﯾﯽ ﻟﻮﺑﯿﺎ

Evaluation of Bio-control and Colonization Potential of Molecular Improved Trichoderma harzianum Strains gainst Rhizoctonia solani Diseases of Bean

ﭼﮑﯿﺪه ﮐﻨﺘﺮل ﺑﯿﻮﻟﻮژﯾﮏ ﯾﮑﯽ از راﻫﮑﺎرﻫﺎي ﻣﺆﺛﺮ ﮐﻨﺘﺮل ﺑﯿﻤﺎريﻫﺎي ﮔﯿﺎﻫﯽ و ﻋﺎﻣﻠﯽ ﺑﺮاي ﮐﺎﻫﺶ ﻣﺼﺮف ﺳﻤﻮم ﺷﯿﻤﯿﺎﯾﯽ اﺳﺖ. ﻗﺎرچ ﺗﺮﯾﮑﻮدرﻣﺎ ﺑﺎ داﺷﺘﻦ ﺧﺎﺻﯿﺖ ﺿﺪﯾﺘﯽ ﻋﻠﯿﻪ ﺑﺴﯿﺎري از ﻗﺎرچﻫﺎي ﺑﯿﻤﺎريزاي ﮔﯿﺎﻫﯽ از ﻣﻮﻓﻖﺗﺮﯾﻦ ﻋﻮاﻣﻞ ﮐﻨﺘﺮل ﺑﯿﻮﻟﻮژﯾﮑﯽ ﻣﺤﺴﻮب ﻣﯽﺷﻮد. ﺗﻮان ﺑﯿﻮﮐﻨﺘﺮﻟﯽ اﯾﻦ ﻗﺎرچ، ﺑﺎ ﻣﯿﺰان ﺗﺠﺰﯾﻪ آﻧﺰﯾﻤﯽ دﯾﻮاره ﺳﻠﻮﻟﯽ ﭘﺎﺗﻮژن، ارﺗﺒﺎط ﻣﺴﺘﻘﯿﻢ دارد. در ﭘﺮوژه ﻗﺒﻠﯽ ﻧﻮﯾﺴﻨﺪﮔﺎن ﺑﻪ ﮐﻤﮏ ﻣﻬﻨﺪﺳﯽ ﭘﺮوﺗﺌﯿﻦ، ﮐﯿﺘﯿﻨﺎز ﮐﺎﯾﻤﺮ 42 ﺳﺎﺧﺘﻪ ﺷﺪ و ﭘﺮوﺗﺌﯿﻦ ﻧﻮﺗﺮﮐﯿﺐ ﺣﺎﺻﻞ ﺑﻪ ﻗﺎرچ T. harzianum اﻧﺘﻘﺎل ﯾﺎﻓﺖ. در ﺗﺤﻘﯿﻖ ﺣﺎﺿﺮ ﺗﻮان ﺑﯿﻮﮐﻨﺘﺮﻟﯽ و ﮐﻠﻨﯿﺰاﺳﯿﻮن ﺟﺪاﯾﻪﻫﺎي ﻣﻬﻨﺪﺳﯽ ﺷﺪه در ﺑﺮاﺑﺮ ﭘﺎﺗﻮژن R. solani در ﺷﺮاﯾﻂ آزﻣﺎﯾﺸﮕﺎه و ﮔﻠﺨﺎﻧﻪ ﺑﺮ روي ﮔﯿﺎه ﻟﻮﺑﯿﺎ ارزﯾﺎﺑﯽ ﺷﺪﻧﺪ. ﭘﺲ از ﺗﺄﯾﯿﺪ ﻣﻮﻟﮑﻮﻟﯽ، ﺣﻀﻮر و ﺛﺒﺎت ﻗﻄﻌﺎت ﻣﻬﻨﺪﺳﯽ ﺷﺪه در رﯾﺴﻪ ﻗﺎرچﻫﺎي ﻣﻬﻨﺪﺳﯽ ﺷﺪه ﺑﻌﺪ از ﮔﺬﺷﺖ دو ﺳﺎل، ﺑﺮرﺳﯽ ﺗﻮان ﺑﯿﻮﮐﻨﺘﺮﻟﯽ در ﮐﺸﺖ ﻣﺘﻘﺎﺑﻞ ﺗﺮﯾﮑﻮدرﻣﺎ و ﭘﺎﺗﻮژن اﻧﺠﺎم ﺷﺪ. ﯾﺎﻓﺘﻪﻫﺎ ﻧﺸﺎن داد ﮐﻪ ﺟﺪاﯾﻪ Chit42- ChBD3 ﺑﺎ 53/82 درﺻﺪ ﺑﺎزدارﻧﺪﮔﯽ ﺑﯿﺶﺗﺮ ﻧﺴﺒﺖ ﺑﻪ ﮐﻨﺘﺮل، ﺑﻬﺘﺮﯾﻦ ﺑﯿﻮﮐﻨﺘﺮل در ﺷﺮاﯾﻂ In vitro ﺑﻮده اﺳﺖ. ﻃﺒﻖ ﻧﺘﺎﯾﺞ ﺑﻪدﺳﺖ آﻣﺪه از آزﻣﺎﯾﺶ ﮔﻠﺨﺎﻧﻪاي ﮔﯿﺎﻫﺎن ﺗﯿﻤﺎر ﺷﺪه ﺑﺎ ﭘﺎﺗﻮژن، ﺟﺪاﯾﻪﻫﺎي ﻣﻬﻨﺪﺳﯽ ﺷﺪه Chit42- ChBD7 ،Chit42- ChBD3 وChit42- ChBD15 در ﺗﻤﺎﻣﯽ ﻣﺮاﺣﻞ ﻣﺨﺘﻠﻒ اﻧﺪازهﮔﯿﺮي دو ﺑﺮﮔﯽ، اواﺳﻂ دوره روﯾﺸﯽ، اواﯾﻞ ﻣﺮﺣﻠﻪ زاﯾﺸﯽ و در ﻣﺮﺣﻠﻪ ﺑﺮداﺷﺖ( ﻋﻼﯾﻢ ﺑﯿﻤﺎري ﮐﻢﺗﺮاز 40 درﺻﺪ را ﻧﺸﺎن دادﻧﺪ. ﮐﻠﻨﯿﺰاﺳﯿﻮن ﺳﻄﺤﯽ رﯾﺸﻪ و ﻫﻢﭼﻨﯿﻦ ﻧﻔﻮذ رﯾﺴﻪ ﻗﺎرچ ﺑﻪ ﻓﻀﺎي ﺑﯿﻦ ﺳﻠﻮﻟﯽ اﭘﯿﺪرم رﯾﺸﻪ ﻟﻮﺑﯿﺎ ﺑﻪوﺳﯿﻠﻪ ﻣﻄﺎﻟﻌﺎت ﻣﻮﻟﮑﻮﻟﯽ ﺑﺎ اﺳﺘﻔﺎده از آﻏﺎزﮔﺮﻫﺎي PF1/R3xba1 ﻣﻮرد ﺗﺎﺋﯿﺪ ﻗﺮار ﮔﺮﻓﺖ.

Biological control of plant diseases is one of the effective strategies to reduce harm effects of pesticides on human health and environment. T. harzianum is one of the most successful biological control agents which have antagonistic activity against many of pathogenic fungi. Biocontrol activity of T. harzianum is linked to the amount and type of hydrolase enzymes. In the authors' previous work, chimeric chitinase was constructed by protein engineering and the recombinant protein was transferred to the T. harzianum. In the present study, the biocontrol activity and colonization of improved isolates were evaluated on bean plant and its pathogen (Rhizoctonia solani) in laboratory and greenhouse. The presence and stability of the chimeric protein was confirmed by molecular technique, after that biocontrol assay was done in dual culture method. The results showed that T3 with 82/53 percent inhibition compared to control, was the best biocontrol in invitro. According to the results of greenhouse tests, plants that were treated with T7, T3 and T15 in all stages of measurement (two leaf, mid-term growth, early reproductive stage and harvest stage) showed symptoms less than 40%. Root surface colonization and fungal hyphae penetration was confirmed by molecular studies using primers PF1/R3xba1.