| Bacterial wilt of plants,caused by Ralstonia solanacearum,is one of the most devastating plant diseases widely distributed in tropical,subtropical,and some warm temperate regions of the world,and has gradually spread to the high latitude,cool areas.R.solanacearum,which causes great economic losses world-wide,has an unusually wide host range since 450 species belonging to 54 botanical families have proved to be affected by the pathogen.Several plant pathogenic bacteria have been reported as potentially causing disease to mulberry,among which,bacterial wilt of mulberry caused by R.solanacearum phylotype I mulberry strains is the most destructive one.Given its economic and scientific importance,R.solanacearum was listed as one of the top ten phytopathogenic bacteria by Molecular Plant Pathology,the famous international journal.A great many research areas concerning genomics,phylogeny,pathogenicity mechanism,detection technique and control method of R.solanacearum have been carried out by researchers throughout the world.At present,13 complete genome sequences of R.solanacearum strains such as GMI1000,Po82,PS107,UY031 and so on have been published and more than 50 draft genome sequences of R.solanacearum strains such as CMR15,FQY4,B50 and so on have been available in NCBI.Broad host range,wide geographical distribution,strain variation,scarcity of host resistance and lack of economically feasible chemical treatments make it difficult to effectively control bacterial wilt disease in the world.Acurate,sensitive and efficient method for detection of R.solanacearum at both species and intra-species strain level is not only profound for disease prevention,but the starting point for the disease early warning system-based management strategy making as well.To explore natural compounds with inhibitory activity to R.solanacearum from plant materials and to develop plant-based pesticides can provide novel ideas for the prevention and control of bacterial wilt,and help to meet the need of the public's major concerns for food,ecology and public health safety.In this paper,we have developed a species-specific LAMP assay for R.solanacearum strains,and a subspecies-specific LAMP assay for R.solanacearum phylotype I mulberry strains.These two assays could quickly and accurately detect R.solanacearum strains and R.solanacearum phylotype I mulberry strains,respectively,from diseased plants sampled in the field.In addition,we have selected four essential oils with inhibitory activity to R.solanacearum and evaluated their antibacterial effects on R.solanacearum in vitro studies,which is expected to get a new method for the control and prevention of bacterial wilt.1.Based on the species-specific gene lpxC of R.solanacearum,four specific LAMP primers were designed and a LAMP assay for rapidly detecting R.solanacearum was established.Visual assessment of DNA amplification by LAMP could be obtained within 1 h.Single-factor experiments were conducted to optimize the parameters of the reaction system,the concentrations of Mg2+ was 6 mmol·L-1,the concentration ratio of inner and outer primers was 8?1?1.6?0.2 ?mol·L-1?and the reacting temperature was determined as 63?.The result of specificity test showed that only the reaction liquids with the DNA of R.solanacearum change green,which indicated this method had a good specificity.Sensitivity experiments indicated that LAMP could detect original DNA,101,102,103,104,and 105 times of diluent,the sensitivity was 1.42 pg which is 10 times higher than conventional PCR.Also this assay could quickly and accurately detect R.solanacearum from plant tissue suspension,diseased plants as well as infected potato tubers sampled in the field.The LAMP assay established in this study had advantages of high sensitivity,specificity,efficiency and low cost over traditional methods and conventional PCR,the reaction results could be directly observed by naked eyes.All the characteristics of LAMP made it suitable to be widely used in field and grass-roots units.2.Based on the subspecies-specific gene MG67 of R.solanacearum phylotype I mulberry strains,six specific LAMP primers were designed and a LAMP assay for rapidly detecting R.solanacearum phylotype I mulberry strains was established.Visual assessment of DNA amplification by LAMP could be obtained within 20 min.Reactions were carried out at different temperatures ranging from 60 to 65°C using ABI 7500 Real Time PCR,the LAMP assay had the highest amplification efficiency at 64°C,compared with the reactions at other temperatures.The result of specificity test showed that only the reaction liquids with the DNA of R.solanacearum phylotype I mulberry strain change green,which indicated this method had a good specificity.This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA which is 10 times higher than conventional PCR,or 2.2×102 CFU/mL of bacterial cells per 25 ?l reaction volume which is 100 times higher than conventional PCR.Moreover,the presence of plant tissue fluid did not affect the sensitivity.In addition,this assay could quickly and accurately detect R.solanacearum phylotype I mulberry strains from diseased plants sampled from the field.Since it does not require expensive equipment or specialized techniques,this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.3.Evaluating the antibacterial effet of 4 essential oils on R.solanacearum with the inhibition zones,MIC and MBC.It showed the antibacterial activities of pure Peppermint oil,essential oil from Amomum tsao-ko and Lavender oil are similar,with the presence of inhibition zones were 5.90±0.0587 mm,6.90±0.0673 mm and 6.29±0.0761 mm,respectively.And pure Litsea cubeba oil can completely inhibit R.solanacearum.The MIC of Peppermint oil,essential oil from Amomum tsao-ko,Lavender oil and Litsea cubeba oil were 4,4,4 and 1 ?L/mL,respectively.The MBC of Peppermint oil,essential oil from Amomum tsao-ko,Lavender oil and Litsea cubeba oil were 4,8,4 and 1 ?L/mL,respectively.4 essential oils can inhibit R.solanacearum by different ways.The gases emitted from Peppermint oil,essential oil from Amomum tsao-ko,Lavender oil and Litsea cubeba oil also had antibacterial activities against R.solanacearum,with the inhibition zones were 7.80±0.037 mm?8.50±0.057 mm?13.10±0.122 mm and 19.00±0.181 mm,respectively.4.The constituents of Litsea cubeba oil were analyzed using a Thermo Finnigan Trace GC ultra-gas chromatograph equipped with Finnigan Trace DSQ mass spectrometer.And evaluating the antibacterial effets and ways to inhibit R.solanacearum of Litsea cubeba oil through mensurating the growth curves of R.solanacearum in culture mediums with different concentrations of Litsea cubeba oil and fumigation effects.The result of GC-MS showed that there were 29 kinds of chemical components in Litsea cubeba oil,and olefins were the main components of Litsea cubeba oil,which accounted for approximately 40 %.The results of growth curves showed that Litsea cubeba oil with different concentrations have different degrees of inhibition on R.solanacearum.With the increase of concentration,the antibacterial effects were enhanced.R.solanacearum was completely inhibited when the concentrations of Litsea cubeba oil were 0.4,0.8 and 1.6 ?L/mL.After 40 hours of fumigation with Litsea cubeba oil,the absorbances at 600 nm of the culture mediums with different concentrations of Litsea cubeba oil 1,0.75,0.5,0.25,0.125 and 0.0625 ?L/mL were 0.0332±0.0069?0.0540±0.0183?0.0619±0.0069?0.0981±0.0746?1.3744±0.0164 and 1.7317±0.0466,which have significant differences with blank control.After fumigating the soil with Litsea cubeba oil for 3 d,the disease indexes of tomato plants were effectively reduced by 36-78% at 8 d when the concentration of Litsea cubeba oil ranged from 0.10 ?L/mL to 0.50 ?L/mL,which indicated that Litsea cubeba oil can be used as a fumigant for soil fumigation of R.solanacearum. |
PDF Full Text Request