Isolation And Identification Of Toxin Produced By Potato Rhizoctonia Solani AG-3 And The Pathogenic Mechanism For Stem Canker

Potato?Solanum tuberosum L.?production has been associated with soil-borne pathogens,and Rhizoctonia solani,a world-wide fungus is found in almost all potato growing-areas of the world,particularly in northwest China,which leads to drastic production losses.The phytotoxins produced by R.solani is one of the most severe pathogenic factors associated with Rhizoctonia disease of potato?stem canker/black scurf?.Although many studies have been conducted on isolation and characterization of R.solani under various soil and climatic conditions in different regions and countries,few experiments have been conducted to characterize R.solani under surroundings in the arid northwest of China,particularly Gansu Province where R.solani is prevalent.This study centered on isolation,characterization of R.solani phytotoxin and the mechanism involve in causing stem canker on potato using various methods?Fourier-transform infrared spectroscopy,and nuclear magnetic resonance?.The study offers contributions in three major ways.The procedures and contributions in all the phases are briefly described below:?1?Isolation,purification and characterization of phytotoxins produced by Rhizoctonia solani AG-3,the causal agent of potato stem canker:In this aspect,a study was conducted to isolate and characterize the phytotoxins extracted from culture filtrates of R.solani isolate anastomosis 3?AG-3?.The culture filtrate was isolated using preparative thin layer chromatography?TLC?method.Fourier-transform infrared spectroscopy?FTIR?,high performance liquid chromatography?HPLC?and nuclear magnetic resonance?NMR?spectral techniques were used to characterize the phytotoxins.Eight toxin fractions were collected by column layer chromatography but only four spots were obtained in the TLC analysis.All the four phytotoxin fractions?third,fourth,sixth and seventh fractions?were seen to have phytotoxic activity.The major functional groups and their peaks identified from FTIR spectrum includes 2962.1 cm^-1?-CH₃?,1465.6 cm^-1?-CH₂-?,1727.9 cm^-1?C=O?,1729.8cm^-1?-COOH?and 1124.3 cm^-1?-OH?.Result from HPLC and NMR showed phytotoxic fractions of 3-methylthiopropionic acid?MTPA?and 3-methylthioacrylic acid?MTAA?with lower molecular weights.The results of this investigation add to the body of evidence that multiple related compounds are involved in R.solani disease development.?2?Investigation of 3-methylthiopropionic acid produced by Rhizoctonia solani AG-3 and its role in the pathogenicity of the fungus:In this contribution,the aim of the study was to investigate the role of 3-methylthiopropionic acid?MTPA?in Rhizoctonia disease using isolates of R.solani AG-3 on potato and determine the concentration at which MTPA will induce disease symptoms on potato in the absence of the fungus.The experiments were laid out in a completely randomized design with five treatments and five replications.The treatments were 0,1,2,4 and 8 mM concentrations of MTPA.Results showed that the MTPA phytotoxin reduced plant growth and tuber yield,compared to the control.Plants treated with 8.0 mM MTPA significantly reduced aboveground fresh weight and belowground fresh weight by 260.14%and 395.95%,respectively,comparative to the control.Plants treated with 8.0 mM MTPA significantly reduced tuber yield by 419.12%compared with the control.Application 8.0 mM MTPA also significantly decreased fresh weight of individual tuber by 100.22%in comparison with control plants without MTPA phytotoxins.The results showed that plants treated with 8.0 mM MTPA also increased biomass of potato plants by 112.44%.The potato seedlings treated with MTPA concentrations of 1.0–8.0 mM induced necrosis of up to 80%of root system area.Cankers were seen on the of potato seedling stems treated with 3-methylthioproprionic acid with 8.0mM MTPA producing the highest stolon and stem canker of 97.78%and 97.10%,respectively.Compared with untreated control,black scurf incidence of potato plants treated with the phytotoxin at a rate of 8 mM was increased by 94.59%.The study demonstrated that R.solani phytotoxin can induce stem canker and black scurf development in the absence of the fungus.?3?Investigations of the effects of R.solani phytotoxins on antioxidant enzyme activities of potato in causing stem canker:In this aspect,the relationship between antioxidant enzyme activities of potato and stem canker was studied.Sample?400 mg?of roots,stems and leaves were separately crushed and ground to a fine powder in a mortar under liquid nitrogen.One portion of powder was suspended in 50 mM^-1 potassium phosphate buffer?PBS?,pH 7.5,in a final volume of 5 ml.The suspension was homogenized and centrifuged.The supernatant was diluted two times and remained frozen in liquid nitrogen and subsequently stored at-70?C for analysis.All of these operations were carried out at 0-4?C.The levels of the various antioxidant enzymes activities and MDA content in the plant extracts were determined spectrophotometrically.Results showed that compared with control plants?CK1?,the activities of antioxidases?superoxide dismutase,peroxidase,and catalase?in potato stems treated with 8 mM MTPA increase by 86.95,203.64,and123.28%,respectively.Similarly,the activity of chitinase in the roots,stems and that of leaves increase by 120.22,145.45,and 143.91%,respectively.However,the activity of malondiadehyde?MDA?in the roots,stems and the leaves of 8 mM MTPA-treated plants decreased by 167.80,156.52,158.30%,respectively,comparative to CK1.The results revealed that phytotoxins and antioxidant enzymes are involved in the disease mechanism of R.solani.In conclusion,FTIR,HPLC,and NMR techniques were successfully used to identify the phytotoxins of R.solani AG-3 from Gansu sites that supply the processing potato industry.The toxic compounds were identified as 3-methylthiopropionic acid?MTPA?and3-methylthioacrylic acid?MTAA?.The study revealed that stem canker increased with increasing amounts of MTPA and MTAA.MTPA increased the activities of the anti-disease enzymes of the potato plant,resulting in exacerbating damage to cellular components due to the higher levels of ROS.The outcome of the entire study is therefore hoped to contribute to the role of the molecular basis of disease resistance against fungal pathogens and production of potato plants with increase disease resistance.It will also enable plant breeders to come out with resistant variety to detoxify the phytotoxins and could provide potato farmers a cheaper and environmentally friendly option in managing stem canker and black scurf caused by Rhizoctonia solani.The work reported here concerning the isolation,characterization of R.solani phytotoxins,and a better insight of the modes of action of toxic compounds may open new,fascinating ways of changing plant genotypes through the use of appropriate techniques.However,it is recommended that further studies on metabolic detoxification of R.solani should be carried in order to assess the performance of the phytotoxin.