| Inner Mongolia is an important potato breeding region and commodity potato production and processing base in China.The expansion of planting areas and continuous cropping obstacles have resulted in increased severity of potato soil-borne diseases,especially potato Verticillium wilt,consequently becoming a major factor limiting potato production.Potato wilt can be prevented and controlled through the extensive use of various chemical agents and soil fumigation.However,this strategy is expensive and can pollute the environment and threaten human health.The cultivation of disease-resistant varieties is crucial,but has not yet been successful.Therefore,exploring efficient and environment-friendly prevention and control measures for potato wilt is particularly important.Previously,a weakly virulent fungal isolate Gibellulopsis nigrescens Vn-1 was isolated from sunflowers and demonstrated that this strain could induce resistance to Verticillium wilt in sunflowers.The differences between the transcriptomes and metabolomes of sunflowers after inoculation with Vn-1 and the highly virulent isolate Vd33 were analysed.This study first established an experimental system for screening the virulence of potato wilt pathogens using soilless cultivation.Through this experimental system,the isolate Vd-36 with the strongest virulence was selected,and the resistance of potatoes induced by the weakly virulent isolate Vn-1 was studied.In order to reveal the induced resistance mechanism of Vn-1 against Vd-36 in potato,a novel protein elicitor was purified from the metabolic fluid of Vn-1.The elicitor can induce potato resistance to the attack of Vd-36 by mainly activating the SA signalling pathway and related defense enzyme activity,and high expression of defense genes,achieving potato resistance to Verticillium wilt.The research results reveal a new mechanism of plant resistance induced by weakly virulent isolates.This provides theoretical guidance for the biological control of potato wilt and the green development of the industry.The main research findings are as follows:1.There are six major findings of this study.First,differences in the pathogenicity of potato Verticillium wilt were compared using soilless,potted,and field cultivation systems.Soilless cultivation for the detection of potato Verticillium wilt pathogenicity was not only as reliable as the potted and field cultivation methods,but significantly superior to the other two methods.The ceramic particles used in soilless cultivation are recyclable,clean,and simple to use.Furthermore,soilless cultivation for pathogen pathogenicity testing has an appreciably shorter experimental period of 30 days,compared to 54 days for pot cultivation and 84 days for field cultivation.The biological colonisation of the inoculated isolates in the roots of the susceptible potato variety Favorite and resistant variety Kexin 1were measured and analysed in the three cultivation systems.The findings demonstrated that soilless cultivation for pathogen pathogenicity testing can effectively reflect the resistance of the variety and is not affected by the environment or other microorganisms.2.The optimal concentration for potato Verticillium wilt resistance induced by Vn-1was 1×10~6 spores/m L and the optimal induction interval was five days.The treatment group(Vn-1+Vd-50)experienced a large increase in reactive oxygen species levels at 12hours post-infection(hpi),and the hydrogen peroxide content reached its peak.In addition,the activities of catalase,superoxide dismutase,and peroxidase(excluding phenylalanine ammonia-lyase),which are enzymes related to plant defence,were greatest at 12 hpi.The salicylic acid(SA)content in leaves of treated potatoes(Vn-1+Vd-50)was higher than that in the control group at 0,12,and 24 hpi,but not at 48 hpi.The SA content was highest at12 hpi,and decreased at 24 hpi,while the St NPR1,St PR2,St PR5,and St PAL2 genes were upregulated.At 24 hpi,the relative St PR1b expression was upregulated.The findings indicate that Vn-1 may activate the biosynthesis and signalling pathways of SA and the relative expression levels of related defence genes,thus preventing potato attack by the virulent isolate Vd-36.3.A novel protein elicitor was isolated and purified from the fermentation broth of the black rotifer Vn-1 using ammonium sulphate precipitation,(?)KTA?protein purification,SDS-PAGE resolution,excision of protein from polyacrylamide gel,mass spectrometry,and other analysis methods.The amino acid sequence of the protein was determined using mass spectrometry.The NCBI’s p BLAST comparison revealed the novel function and structure of the protein elicitor.The 17 k D protein elicitor induced(HR)reactions in tobacco and potato leaves.An 18 amino acid signal peptide was present at the N-terminal of the elicitor.Prediction analysis of its secondary structure revealed that the monomer of the protein activator contains twoαspirals,six reverse parallel folds,and multiple irregular curls.Homologous modelling and analysis of the tertiary structure revealed a groove in the protein monomer that is prone to dimer formation.4.The protein elicitor Pe GD1 was recombined and expressed upon isopropylβ-d-1-thiogalactopyranoside(IPTG)induction.The complete coding sequence of 405-bp Pe GD1,obtained by PCR amplification using specific primers.Pe GD1 was cloned and ligated in the T cloning vector p MD19-T.The p RSET-CH-Pe GD1 vector was constructed by enzymatic digestion,linking,and other methods.After verification,the plasmids were transferred into Escherichia coli BL21 cells.Following IPTG induction,Pe GD1 was expressed in large quantities.5.Protein elicitor may exhibit host selectivity.Interestingly,24 h after injection of different concentrations of the protein elicitor into tobacco and potato leaves,HR reactions were evident only in tobacco leaves at an elicitor concentration of 1000μg/m L.In potato leaves,as the concentration of the elicitor increased,the HR reaction gradually strengthened.Measurements at different time points of the SA and jasmonic acid/ethylene(JA/ET)content and the relative expression of related synthetic genes in potato leaves inoculated with Vd-36,24 h after injecting Pe GD1 into stems,revealed that Pe GD1induced systemic acquired resistance in potatoes and activated the SA signalling pathway at 12 hpi,while weakly activating the JA/ET signalling pathway at 24 hpi,producing resistance of potatoes to Vd-36.The relative expression levels of plant pathogenesis-related resistance gene St PR1b were significantly higher than those of the control at all time points,except 0 hpi,indicating that this gene plays a major role in the resistance of potatoes to pathogens.6.Three conserved sites of the St PR1b gene were silenced using the virus-induced gene silencing technology,resulting in a total of 300 bp silenced plants.After pre-treatment of the silenced plants with Pe GD1 buffer and inoculation with the highly virulent V.dahliae Vd-36,the symptoms of Verticillium wilt in the silenced plants were significantly reduced than those in plants inoculated with Vd-36 alone,suggesting that the St PR1b gene plays a major role in resistance to pathogens in potatoes. |
PDF Full Text Request