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1、Control of Bacterial Wilt of Geranium with Phosphorous Acid D.J.Norman 磷酸对天竺葵青枯病的控制,主要内容：,一：前言二：材料与方法三：结果与讨论,一、前言,Once it becomes established in a susceptible crop,southern bacterial wilt,caused by Ralstonia solanacearum is very hard to eradicate.There are two primary avenues by which R.solanacearum
2、 moves and gains access to crops:one is via water,and the other is through infected propagation materials.由青枯菌引起的南方青枯病一旦侵染敏感作物，就很难根除。青枯菌侵染作物的两种途径是：通过水流侵染通过被侵染物质传播。To combat bacterial wilt,resistant cultivars have been recommended in areas of the world where the pathogen is endemic.In susceptible gre
3、enhousegrown crops,only strict sanitation has been successful in prohibiting plant infestation.为了抵抗地方性青枯病，世界上很多地方都推荐种植抗性作物。种植在温室的敏感作物只有控制环境卫生才能抑制其侵染。,Geraniums are susceptible to races 1 and 3 of R.solanacearum.Most geraniums produced in the world are vegetatively propagated in Guatemala,Costa Rica,
4、Columbia,China,and Kenya.In all these locations,endemic populations of R.solanacearum exist.There are no known treatments that are effective in protecting geranium plants.天竺葵对于青枯菌属1和3是敏感的，天竺葵广泛种植在世界各地如危地马拉，哥斯达黎加，哥伦比亚，中国和肯尼亚。但这些地方都存在地方性青枯病，目前还没有有效的方法保护天竺葵作物。Thus,the objective of this research was to
5、determine if geranium plants could be protected from infection with applications of selected bactericides and chemicals.因此本实验的目的就是对天竺葵作物施以筛选过的细菌和化学药品来验证是否能保护作物免受侵染。,二、材料与方法1 Screening of products.,A race 1(biovar 1)strain(R1B1)isolated in Florida(P673)from pothos(Epipremnum aureum(Linden&Andre)Blunt
6、.)was used in the initial screening of prospective products.The isolates from pothos are of Central American origin and possess a broader host range than strains endemic in Florida.在弗罗里达的黄金葛里分离出来的R1B1（P673）被用于开始预期产品的筛选。它是在在美国中部分离出来的菌属比弗罗里达的地方性菌属更原始，且具有更广泛的寄主。Promising products were later tested in e
7、nvironmental chambers with a race 3(biovar 2)strain(R3B2)isolated from geranium(UW551),provided by C.Allen,University of Wis-consin.更多的产品将会用R3B2(UW551)在环境检测箱中进行检测，R3B2有威斯康辛大学的艾伦提供。,材料包括：三异丙基乙黄酰，苯并噻二唑，硫酸铜，氢氧化铜，铜锡，铜盐，双氧水，糖醛，苯烷基二甲基氯化铵，奥索利酸(Starner)，枯草芽孢杆菌，磷酸钾盐。The products were mixed in water at the hi
8、ghest labeled rates recommended by the manufacturers for plant production.Products are not labeled for control of Ralstonia.These products were applied as a drench through potting medium at 50 ml per 9-cm pot.In the initial screening,products were applied every 7 days for a total of four application
9、s.Plants were inoculated with R.solanacearum 3 days after the first application.Products were not retested unless plants were protected from infection.这些物质以浸液浇到盆栽介质中，每盆50 ml，浇到9cm深处。在开始的筛选中每7天施一次，总共4次。第一次施用之后3天开始接种青枯菌。这些物质不再测定除非作物被侵染。,Products were screened with zonal geranium plants.Rooted geranium
10、 cuttings were transplanted into 9-cm-diameter plastic pots containing 85 g of Vergo Container Mix A(60%Canadian peat,20%vermiculite,and 20%perlite by volume,pH 6.5)and fertilized with 1.5 g of Osmocote 15-9-12 with micronutrients per pot.产品的筛选用带状天竺葵，其根要移栽到直径为9cm的塑料盆中，盆中有85g介质（60%碳泥,20%蛭石,and 20%珍珠岩
11、,pH 6.5）和1.5g奥绿肥（15-9-12）。Plants were grown for a minimum of 4 weeks before treatments.Experiments were conducted in greenhouses with temperatures maintained between 18 and 32C and light levels between 285 and 380 mol.m-2.s-1.Plants were arranged in a completely randomized design with 10 plants per
12、treatment.在处理之前，作物最少生长4周，该实验在温室中进行，温度：18 到 32C，光照：285 到 380 mol.m-2.s-1.实验完全随机区组，每个处理10株。,Two additional treatments were included in each test:a noninoculated control(saline,8.5 g NaCl/liter applied without R.solanacearum)and a disease control(no product applied,inoculated with pathogen).每个实验增加两个处理：
13、不接种控制（含有盐分，NaCl，无青枯菌）;病害控制（不施其他物质，但接种病菌）。For inoculum production,R.solanacearum strains were grown on triphenyltetrazolium chloride(TZC)medium(11)for 48 h,and cells were harvested and spectrophotometrically adjusted(A600)in saline to 5 108CFU/ml.接种青枯菌株的要在氯化三苯基四氮唑（TZC）介质中生长48小时，达到5 108CFU/ml.的时候收获菌株。
14、,Wilt symptoms were recorded as they occurred.Bacteria were reisolated 2 weeks after last treatment(6 weeks after inoculation)from plants without wilt symptoms.A cross-section of stem 1 cm in length was removed approximately 0.5 cm above the soil line of each geranium plant.萎蔫症状一出现就要记录下来，最后一次处理之后两周，
15、细菌就要从没有萎蔫症状的植物中分离。取1cm茎，从离地面0.5cm开始取。通过处理把悬浮液加到TZC介质中培养然后进行检测。2 Further testing of promising products.Geranium plants were found not to be systemically infected by R.solanacearum when treated with either K-Phite(a.i.53%mono-and di-potassium salts of phosphorous acid)or Starner(a.i.20%oxolinic acid)a
16、t the initial test rates.在初始的检测中，当施以53%的磷酸一钾或二钾或 Starner，天竺葵不能被青枯菌系统性侵染。,To determine effective application rates and intervals for these products,four rates were tested(vol/vol,0.25,0.5,0.75,1.0%)at three application regimes(one application;two applications at 14-day interval;and four applications
17、at 7-day interval).Bactericides were applied 3 days before pathogen inoculation as in the initial product screen.为了测定这些物质的有效性和周期，用4种比率（0.25,0.5,0.75,1.0%）测定，用三种施用方式（一次性施用，两次施用间隔为14天，四次施用间隔为7天)，杀菌剂在接种病原菌之前三天施用。Each treatment again contained 10 replicates.Using the R1B1 strain,four tests were conducte
18、d with two tests at each inoculum concentration:5 108 CFU/ml(3 108 CFU/gram soil),and 1 107CFU/ml(6 106 CFU/gram soil).每个处理10个重复，用R1B1菌株，进行了两个接种测试：5 108 CFU/ml(3 108 CFU/gram soil),1 107CFU/ml(6 106 CFU/gram soil).,Two additional controls(five plants each)were added at the lower inoculum rate to mon
19、itor concentrations of R.solanacearum within the potting medium.These controls consisted of:(i)pots containing potting medium without a geranium plant,yet inoculated with R.solanacearum,and(ii)pots containing potting medium without plant or R.solanacearum inoculation.Additional controls were watered
20、 like all other treatments.在接种较低的盆栽介质中增加了两个处理，每个处理5株，只有盆栽介质(盆栽介质+天竺葵，盆栽介质+接种)。与其它处理一样浇水。Six weeks after first chemical application,a cork borer was used to retrieve a 1-g sample of potting medium from the first five replications of all treatments.A dilution series of the soil sample was done in SDW
21、and replicaplated onto modified semiselective medium.,Typical R.solanacearum colonies were counted after 48 h growth at 28C.Bacteria were reisolated from all geranium plants 6 weeks after inoculation,as previously described.Colonies from plates with typical R.solanacearum growth pattern were suspend
22、ed in saline(8.5 g/liter NaCl)and injected into parenchymatous tissue of tobacco for hypersensitive response(HR)following protocols of Lozano and Sequeira(12).在28C下培养48小时后计算青枯菌群，接种6周后从天竺葵中分离细菌，如前面所述得到的悬浮液注入马铃薯的薄壁组织中来测其敏感性。分离茎和土壤，把马铃薯种到每个盆子中来验证青枯菌是否足够病害发展。马铃薯对于R1B1 和R3B2菌属是十分敏感的，最少4周后就会出现症状，与前面相同，出现症
23、状就进行分离。,The R3B2 strain was tested in environmental chambers set on a 12-h day/night cycle(19C night,24C day,310 mol.m-2.s-1).Two tests were conducted at an inoculum concentration of 5 108CFU/ml(3 108 CFU/gram soil).All other experimental parameters and controls were the same as previously described
24、.R3B2在环境检测箱内放12h后测定，两个实验的接种浓度为5 108CFU/ml 其它的实验参数和控制条件都与前面相同。Antibacterial efficacy of compounds containing phosphorus Many products containing P are currently sold in the ornamental plant industry as fertilizers or fungicides.Common ingredients include P2O5,H3PO3,and H3PO4 with mono-and/or di-potas
25、sium.We tested the efficacy for controlling bacterial wilt by P2O5,H3PO3,H3PO4,and KCl.很多含磷产品在工厂都以肥料或菌剂来买，一般有P2O5,H3PO3,kH2PO4，k2HPO4。我们用P2O5,H3PO3,H3PO4,和 KCl来测它们对青枯菌的控制效率。,Reagent grade KCl and the three P chemicals buffered with CaCO3 were used in the first test,and the P chemicals only buffered
26、with KOH were used in the sec-ond test.We used four concentrations of P,0.032,0.063,0.095,and 0.127%,for each of the three chemicals in the two tests;and the first four concentrations of K,0.054,0.108,0.162,and 0.216%,were made from KCl.第一个实验中，用CaCO3来缓冲KCl和三种含磷化合物。第二个实验中用KOH来缓冲含磷化合物，四种P的浓度为 0.032,0.
27、063,0.095,0.127%,两个处理的三种含磷物质浓度一样，4种K的浓度为0.054,0.108,0.162,0.216%。As in the initial product,screening solutions were applied on a 7-day interval with four applications.Again,plants(10 per replication)were inoculated with strain R1B1 3 days after the first compound application.Two weeks after last app
28、lication,all plants were sampled for R.solanacearum as previously described.在起始筛选中，筛选溶液将会以7天的间隔施用在四个处理中。作物接种R1B1菌，三天后施用第一次，最后一次施用之后两周收集所有植株来详细描述青枯菌。,We measured the in vitro effect of phosphorous acid on the growth of both R1B1 and R3B2 strains.Flasks containing 40 ml of the following solutions were
29、 used:(i)SDW,(ii)SDW+P at 0.127%as H3PO3,(iii)SDW+K-Phite at 1%,(iv)Nutrient broth(Difco)containing 5 g sucrose(NB),(v)NB+P at 0.127%as H3PO3,and(vi)NB+K-Phite at 1%.我们在试管中进行了磷酸对 R1B1 和R3B2的控制实验，分6组。分别为灭菌水灭菌水+0.127%H3PO3灭菌水+1%磷酸钾营养液营养液+0.127%H3PO3营养液+1%磷酸钾 Concentrations of viable cells in each flas
30、k were enumerated on 0,1,7,14,21,and 28 days.Counts were discontinued when concentrations of cells reached 109CFU/ml.在适当条件下进行培养，在0,1,7,14,21,和28 天计算其存活的细菌数量。当其浓度达到109CFU/ml，不在计算。,Safety of K-Phite to crop.,To determine if phytotoxicity occurred with the K-Phite product,an experiment was set up as pr
31、eviously described using four concentrations(vol/vol,0.50,0.75,1,and 2%)with 10 plants per treatment.Dry weights of both roots and foliage of all plants were determined at the end of the test.In addition,we measured medium pH and soluble salts for five plants of each treatment and performed a comple
32、te foliar analysis.为了确定K-Phite 对作物是否安全，我们进行了如前面所述4个浓度（0.50,0.75,1,和2%），每个处理10株。实验最后测所有植株的根系和叶片，每个处理测5株的介质浓度和盐溶液。对叶片进行完整的分析。,RESULTS,Screening of products.Further testing of promising products.Efficacy of phosphorous compounds.Safety of K-Phite to crop.,Screening of products.In the initial testing,ne
33、arly all the products slowed disease progress.However,they did not protect the plants from infection,except for benzothiadiazole(Actigard),oxolinic acid(Starner),and potassium salts of phosphorous acid(K-Phite)(data not shown).Further testing of benzothiadia-zole was discontinued due to leaf absciss
34、ion.This abscission occurred even at rates as low as 5 l/liter.在开始的测试中，所有的物质都能减慢病害发生的过程，但不能保护作物免受侵染，除了苯并噻二唑，奥利索酸和磷酸钾盐。而苯并噻二唑即便在很低的浓度下也会损害叶片，所以不再继续实验。Further testing of promising products.Low rates and intervals of Starner were ineffective in protecting plants from infection.High rates above 0.5%on a
35、 7-day interval provided protection for the majority of the plants.However,at inoculation rates:of 3 108 CFU/gram soil and 6 106 CFU/gram soil,systemic infections were occasionally observed in plants treated with 0.75 and 1%Starner.Due to the high rates and intervals required for disease protection,
36、testing of Starner was discontinued.低比率和间隔的Starner没有保护作用，高于0.5%虽有效，但作物还是会出现偶然的系统性侵染，所以对Starner不再进行实验。,K-Phite was fairly effective in protecting geranium plants from infection at 3 108CFU/gram soil(Fig.1)and very effective in protecting plants at 6 106 CFU/gram soil.在接种浓度为 3 108CFU/gram时，磷酸钾的保护作用是有效
37、的，在 6 106 CFU/gram 时保护效果非常明显。具体情况如图一所示。,Populations of R.solanacearum were not detectable in the soil after potting medium was drenched with K-Phite for either two or four applications(Fig.2).the R1B1 strain was more persistent than the R3B2 strain while the R3B2 strain was undetectable at that time
38、.在两次或四次施用磷酸钾的处理中检测部到青枯菌。R1B1比 R3B2更持久，R1B1在接种后6周还可以检测到青枯菌但R3B2。,Fig.1.Effects of K-Phite application on control of Ralstonia solanacearum infection.,Efficacy of phosphorous compounds.K-Phite effectively protected geranium plants from infection with the R1B1 strain at all four applied rates(Fig.3).Si
39、nce it is a product of potassium salts of phosphorous acid,three P chemicals,P2O5,H3PO3,and H3PO4,were tested.如图3所示：磷酸钾能的4个处理都能保护作物免受侵染，而其它含磷物质却不可以。,Fig.3.Relative effects of K-Phite and phosphorus compounds in control of Ralstonia solanacearum(strain P673,R1B1).,The long-term in vitro growth curves
40、 were similar for both bacterial strains tested.In Figure 4,results are displayed for UW551.Phosphorous acid either in reagent grade or product form inhibited cell replication in NB and SDW(Fig.4).两种细菌的长期试管生长曲线都相似，R3B2菌属结果如图所示。Safety of K-Phite to crop.There was a significant(P=0.5)increase in the g
41、eranium shoot dry weight of 27%with the 1%K-Phite treatment relative to the water control.All other shoot dry weights were not significantly different from the water control.Iron levels decreased from 122.5 ppm to 45.7 ppm(R2 0.58).Leaves were less green on plants treated with 1%K-Phite than on plan
42、ts treated with waterIn order to maintain healthy geranium plants,the K-Phite product should not be drenched at rates above 1%on a prolonged basis。与空白对照相比，用1%磷酸钾处理的天竺葵地上部干重明显增加了27%，别的处理与空白对照无显著性差异。但磷酸钾高于1%时，叶片中铁的含量从122.5 ppm减低到45.7 ppm，叶片发黄。所以磷酸钾的比率不能高于1%。,Fig.4.Growth of Ralstonia solanacearum UW55
43、1 in flasks containing 40 ml of the following solutions:()sterile distilled water(SDW),()SDW+0.127%P as H3PO3,()SDW+1%K-Phite(53%mono-and di-potassium salts of phosphorous acid),()Nutrient broth Difcocontaining 5 g sucrose(NB),()NB+0.127%P as H3PO3,and()NB+1%K-Phite.,DISCUSSION,The majority of the g
44、eraniums sold in the global north,but where R.solanacearum is endemic.In order to exclude R3B2 from these geranium production acilities,rigorous sanitation measures are being implemented(24).However,complete eradication of bacterial wilt is difficult given an environment conducive to both host and p
45、athogen.Thus,in addition o sanitation and resistant cultivars,growers need other control options.天竺葵的主要产地是北半球，但存在地方性青枯病，为了控制天竺葵作物R3B2菌，对生产设施进行了严格的控制，然而一般很难完全根除。因此，除了控制环境和栽培抗性品种，种植者也需要其他控制。,This study showed that most of the tested products slowed the progression of bacterial wilt on geranium,but did
46、 not protect the plants from infection and subsequent death.Only phosphorous acid protected plants from infection.It appears that the protection occurs in the soil and root matrix,as pathogen cells could not be cultured from the potting medium of treatments with high frequency and application rates
47、of K-Phite.本实验表明，大多数检测产品都能减慢青枯菌在天竺葵中的传播过程，但只有磷酸才能保护天竺葵免受侵染。在高频率和高比率施用磷酸钾的处理中，分离不出病菌，所以保护部位是在土壤和根系。,The mechanism by which phosphorous acid inhibits growth of R.solanacearum is not known.Only phosphorous acid(H3PO3),not phosphorus pentoxide(P2O5)or phosphoric acid(H3PO4),was effective in control of t
48、his pathogen(Figs.2 and 3).One difference among the three compounds is that P in P2O5 and H3PO4 has valence 5 but the valence is 3 in H3PO3.Whether the valence differences made the difference in this pathogen control is unknown.磷酸抑制青枯菌生长的机制尚未清楚，H3PO3，P2O5，和H3PO4。只有H3PO3才能有效抑制病菌生长，这三种化合物的不同的点是P在其中的化合
49、价不同，在H3PO3中是3，别的事5。是否是化合价差异导造成对该病菌控制的差异也善不清楚。,The R1B1 isolate appeared in this study to be far more aggressive and harder to control on geranium than the R3B2 isolate.In addition,the R1B1 isolate survived better than the R3B2 isolate in planting media without the presence of a plant.Broad conclusio
50、ns regarding pathogenicity and survival of these two races of R.solanacearum should not be made from these results.Phosphorous acid does not prevent plant wilt and death when geranium stems are inoculated with low concentrations of R.solanacearum.The effectiveness and safety of the various products
