讀書報告12磷酸對天竺葵青枯病的控制課件

1、Control of Bacterial Wilt of Geranium with Phosphorous Acid D. J. Norman 磷酸對天竺葵青枯病的控制第1頁，共31頁。主要內(nèi)容：一：前言二：材料與方法三：結(jié)果與討論第2頁，共31頁。一、前言O(shè)nce 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 wh

2、ich R. solanacearum 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，就很難根除。青枯菌侵染作物的兩種途徑是：通過水流侵染通過被侵染物質(zhì)傳播。To combat bacterial wilt, resistant cultivars have been recommended in areas of the world where the pathogen is ende

3、mic .In susceptible greenhousegrown crops, only strict sanitation has been successful in prohibiting plant infestation.為了抵抗地方性青枯病，世界上很多地方都推薦種植抗性作物。種植在溫室的敏感作物只有控制環(huán)境衛(wèi)生才能抑制其侵染。第3頁，共31頁。Geraniums are susceptible to races 1 and 3 of R. solanacearum . Most geraniums produced in the world are vegetatively

4、propagated in Guatemala, Costa Rica,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. 天竺葵對于青枯菌屬1和3是敏感的，天竺葵廣泛種植在世界各地如危地馬拉，哥斯達(dá)黎加，哥倫比亞，中國和肯尼亞。但這些地方都存在地方性青枯病，目前還沒有有效的方法保護(hù)天竺葵作物

5、。 Thus, the objective of this research was to determine if geranium plants could be protected from infection with applications of selected bactericides and chemicals.因此本實驗的目的就是對天竺葵作物施以篩選過的細(xì)菌和化學(xué)藥品來驗證是否能保護(hù)作物免受侵染。第4頁，共31頁。二、材料與方法1 Screening of products. A race 1 (biovar 1) strain (R1B1) isolated in Flo

6、rida (P673) from pothos (Epipremnum aureum (Linden & Andre) Blunt.) 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.在弗羅里達(dá)的黃金葛里分離出來的R1B1（P673）被用于開始預(yù)期產(chǎn)品的篩選。它是在在美國中部分離出來的菌

7、屬比弗羅里達(dá)的地方性菌屬更原始，且具有更廣泛的寄主。Promising products were later tested in environmental chambers with a race 3 (biovar 2) strain (R3B2) isolated from geranium (UW551), provided by C. Allen, University of Wis-consin.更多的產(chǎn)品將會用R3B2 (UW551)在環(huán)境檢測箱中進(jìn)行檢測，R3B2有威斯康辛大學(xué)的艾倫提供。第5頁，共31頁。材料包括：三異丙基乙黃酰，苯并噻二唑，硫酸銅，氫氧化銅，銅錫，銅鹽，雙

8、氧水，糖醛，苯烷基二甲基氯化銨，奧索利酸(Starner)，枯草芽孢桿菌，磷酸鉀鹽。The products were mixed in water at the highest 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 t

9、he initial screening, products were applied every 7 days for a total of four applications. Plants were inoculated with R. solanacearum 3 days after the first application. Products were not retested unless plants were protected from infection.這些物質(zhì)以浸液澆到盆栽介質(zhì)中，每盆50 ml，澆到9cm深處。在開始的篩選中每7天施一次，總共4次。第一次施用之后3

10、天開始接種青枯菌。這些物質(zhì)不再測定除非作物被侵染。第6頁，共31頁。Products were screened with zonal geranium plants . Rooted geranium 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

11、of Osmocote 15-9-12 with micronutrients per pot.產(chǎn)品的篩選用帶狀天竺葵，其根要移栽到直徑為9cm的塑料盆中，盆中有85g介質(zhì)（60% 碳泥, 20%蛭石 , and 20% 珍珠巖, pH 6.5）和1.5g奧綠肥（ 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 li

12、ght levels between 285 and 380 mol.m-2.s-1. Plants were arranged in a completely randomized design with 10 plants per treatment.在處理之前，作物最少生長4周，該實驗在溫室中進(jìn)行，溫度：18 到 32C ，光照：285 到 380 mol.m-2.s-1. 實驗完全隨機(jī)區(qū)組，每個處理10株。第7頁，共31頁。Two additional treatments were included in each test: a noninoculated control (sal

13、ine, 8.5 g NaCl/liter applied without R. solanacearum) and a disease control (no product applied, inoculated with pathogen). 每個實驗增加兩個處理：不接種控制（含有鹽分，NaCl，無青枯菌）;病害控制（不施其他物質(zhì)，但接種病菌）。 For inoculum production, R. solanacearum strains were grown on triphenyltetrazolium chloride (TZC) medium (11) for 48 h, a

14、nd cells were harvested and spectrophotometrically adjusted (A600) in saline to 5 108CFU/ml. 接種青枯菌株的要在氯化三苯基四氮唑（TZC）介質(zhì)中生長48小時， 達(dá)到5 108CFU/ml.的時候收獲菌株。第8頁，共31頁。Wilt symptoms were recorded as they occurred. Bacteria were reisolated 2 weeks after last treatment (6 weeks after inoculation) from plants wit

15、hout 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. 萎蔫癥狀一出現(xiàn)就要記錄下來，最后一次處理之后兩周，細(xì)菌就要從沒有萎蔫癥狀的植物中分離。取1cm莖，從離地面0.5cm開始取。通過處理把懸浮液加到TZC介質(zhì)中培養(yǎng)然后進(jìn)行檢測。 2 Further testing of promising products. Geranium plants were found not to b

16、e 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) at the initial test rates. 在初始的檢測中， 當(dāng)施以53%的磷酸一鉀或二鉀或 Starner，天竺葵不能被青枯菌系統(tǒng)性侵染。第9頁，共31頁。To determine effective application rates and

17、 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 at 7-day interval). Bactericides were applied 3 days before pathogen inoculation as in the initial product s

18、creen.為了測定這些物質(zhì)的有效性和周期，用4種比率（0.25, 0.5, 0.75, 1.0%）測定，用三種施用方式（一次性施用，兩次施用間隔為14天，四次施用間隔為7天)，殺菌劑在接種病原菌之前三天施用。 Each treatment again contained 10 replicates. Using the R1B1 strain, four tests were conducted with two tests at each inoculum concentration: 5 108 CFU/ml (3 108 CFU/gram soil), and 1 107CFU/ml

19、(6 106 CFU/gram soil). 每個處理10個重復(fù)，用R1B1菌株，進(jìn)行了兩個接種測試：5 108 CFU/ml (3 108 CFU/gram soil), 1 107CFU/ml (6 106 CFU/gram soil). 第10頁，共31頁。Two additional controls (five plants each) were added at the lower inoculum rate to monitor concentrations of R.solanacearum within the potting medium. These controls c

20、onsisted 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 like all other treatments. 在接種較低的盆栽介質(zhì)中增加了兩個處理，每個處理5株，只有盆栽介質(zhì)(盆栽介質(zhì)+天竺葵，盆

21、栽介質(zhì)+接種)。與其它處理一樣澆水。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 and replicaplated onto modified semiselective medium.第11頁，共31頁。Typic

22、al 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 suspended in saline (8.5 g/liter NaCl) and injected into pare

23、nchymatous tissue of tobacco for hypersensitive response (HR) following protocols of Lozano and Sequeira (12). 在28C下培養(yǎng)48小時后計算青枯菌群，接種6周后從天竺葵中分離細(xì)菌，如前面所述得到的懸浮液注入馬鈴薯的薄壁組織中來測其敏感性。分離莖和土壤，把馬鈴薯種到每個盆子中來驗證青枯菌是否足夠病害發(fā)展。馬鈴薯對于R1B1 和R3B2菌屬是十分敏感的，最少4周后就會出現(xiàn)癥狀，與前面相同，出現(xiàn)癥狀就進(jìn)行分離。 第12頁，共31頁。The R3B2 strain was tested in

24、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.R3B2在環(huán)境檢測箱內(nèi)放12h后測定，兩個實驗的接種濃度為5

25、108CFU/ml 其它的實驗參數(shù)和控制條件都與前面相同。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-potassium. We tested the efficacy

26、 for controlling bacterial wilt by P2O5, H3PO3, H3PO4, and KCl. 很多含磷產(chǎn)品在工廠都以肥料或菌劑來買，一般有P2O5, H3PO3, kH2PO4，k2HPO4。我們用P2O5, H3PO3, H3PO4,和 KCl來測它們對青枯菌的控制效率。第13頁，共31頁。Reagent grade KCl and the three P chemicals buffered with CaCO3 were used in the first test, and the P chemicals only buffered with KOH

27、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. 第一個實驗中，用CaCO3來緩沖KCl和三種含磷化合物。第二個實驗中用KOH來緩沖含磷化合物，四種P的濃度為 0.03

28、2, 0.063, 0.095, 0.127%,兩個處理的三種含磷物質(zhì)濃度一樣，4種K的濃度為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 week

29、s after last application, all plants were sampled for R. solanacearum as previously described. 在起始篩選中，篩選溶液將會以7天的間隔施用在四個處理中。作物接種R1B1菌，三天后施用第一次，最后一次施用之后兩周收集所有植株來詳細(xì)描述青枯菌。第14頁，共31頁。We measured the in vitro effect of phosphorous acid on the growth of both R1B1 and R3B2 strains. Flasks containing 40 ml of

30、 the following solutions were 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%. 我們在試管中進(jìn)行了磷酸對 R1B1 和R3B2的控制實驗，分6組。分別為滅菌水滅菌水+0.127% H3PO3滅菌水+1%磷酸鉀營養(yǎng)液營養(yǎng)液+0.127%

31、H3PO3營養(yǎng)液+1%磷酸鉀 Concentrations of viable cells in each flask were enumerated on 0, 1, 7, 14, 21, and 28 days.Counts were discontinued when concentrations of cells reached 109CFU/ml.在適當(dāng)條件下進(jìn)行培養(yǎng)，在0, 1, 7, 14, 21, 和28 天計算其存活的細(xì)菌數(shù)量。當(dāng)其濃度達(dá)到109CFU/ml，不在計算。第15頁，共31頁。Safety of K-Phite to crop.To determine if ph

32、ytotoxicity occurred with the K-Phite product, an experiment was set up as previously 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

33、 medium pH and soluble salts for five plants of each treatment and performed a complete foliar analysis.為了確定K-Phite 對作物是否安全，我們進(jìn)行了如前面所述4個濃度（ 0.50, 0.75, 1, 和2%），每個處理10株。實驗最后測所有植株的根系和葉片，每個處理測5株的介質(zhì)濃度和鹽溶液。對葉片進(jìn)行完整的分析。第16頁，共31頁。RESULTSScreening of products.Further testing of promising products.Efficacy of

34、 phosphorous compounds. Safety of K-Phite to crop. 第17頁，共31頁。Screening of products. In the initial testing, nearly 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 o

35、f phosphorous acid (K-Phite) (data not shown). Further testing of benzothiadia-zole was discontinued due to leaf abscission. This abscission occurred even at rates as low as 5 l/liter.在開始的測試中，所有的物質(zhì)都能減慢病害發(fā)生的過程，但不能保護(hù)作物免受侵染，除了苯并噻二唑，奧利索酸和磷酸鉀鹽。而苯并噻二唑即便在很低的濃度下也會損害葉片，所以不再繼續(xù)實驗。Further testing of promising p

36、roducts. Low rates and intervals of Starner were ineffective in protecting plants from infection. High rates above 0.5% on a 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 occa

37、sionally observed in plants treated with 0.75 and 1% Starner. Due to the high rates and intervals required for disease protection, testing of Starner was discontinued. 低比率和間隔的Starner沒有保護(hù)作用，高于0.5%雖有效，但作物還是會出現(xiàn)偶然的系統(tǒng)性侵染，所以對Starner不再進(jìn)行實驗。第18頁，共31頁。K-Phite was fairly effective in protecting geranium plant

38、s from infection at 3 108CFU/gram soil (Fig. 1) and very effective in protecting plants at 6 106 CFU/gram soil. 在接種濃度為 3 108CFU/gram時，磷酸鉀的保護(hù)作用是有效的，在 6 106 CFU/gram 時保護(hù)效果非常明顯。具體情況如圖一所示。第19頁，共31頁。第20頁，共31頁。Populations of R. solanacearum were not detectable in the soil after potting medium was drenched

39、 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.在兩次或四次施用磷酸鉀的處理中檢測部到青枯菌。R1B1比 R3B2更持久，R1B1在接種后6周還可以檢測到青枯菌但R3B2。第21頁，共31頁。Fig. 1. Effects of K-Phite application on control of Ralston

40、ia solanacearum infection. 第22頁，共31頁。Efficacy of phosphorous compounds.K-Phite effectively protected geranium plants from infection with the R1B1 strain at all four applied rates (Fig. 3). Since it is a product of potassium salts of phosphorous acid, three P chemicals, P2O5, H3PO3, and H3PO4, were t

41、ested. 如圖3所示：磷酸鉀能的4個處理都能保護(hù)作物免受侵染，而其它含磷物質(zhì)卻不可以。第23頁，共31頁。Fig. 3. Relative effects of K-Phite and phosphorus compounds in control of Ralstonia solanacearum(strain P673, R1B1). 第24頁，共31頁。The long-term in vitro growth curves were similar for both bacterial strains tested. In Figure 4, results are display

42、ed for UW551. Phosphorous acid either in reagent grade or product form inhibited cell replication in NB and SDW (Fig. 4). 兩種細(xì)菌的長期試管生長曲線都相似，R3B2菌屬結(jié)果如圖所示。Safety of K-Phite to crop. There was a significant (P = 0.5) increase in the geranium shoot dry weight of 27% with the 1% K-Phite treatment relative

43、 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 plants treated with waterIn order to maintain healthy geranium plant

44、s, the K-Phite product should not be drenched at rates above 1% on a prolonged basis。與空白對照相比，用1%磷酸鉀處理的天竺葵地上部干重明顯增加了27%，別的處理與空白對照無顯著性差異。但磷酸鉀高于1%時，葉片中鐵的含量從122.5 ppm減低到45.7 ppm，葉片發(fā)黃。所以磷酸鉀的比率不能高于1%。第25頁，共31頁。Fig. 4. Growth of Ralstonia solanacearum UW551 in flasks containing 40 ml of the following solut

45、ions: () 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. 第26頁，共31頁。DISCUSSION The majority of the geraniums so

46、ld 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 pathog

47、en. Thus, in addition o sanitation and resistant cultivars, growers need other control options. 天竺葵的主要產(chǎn)地是北半球，但存在地方性青枯病，為了控制天竺葵作物R3B2菌，對生產(chǎn)設(shè)施進(jìn)行了嚴(yán)格的控制，然而一般很難完全根除。因此，除了控制環(huán)境和栽培抗性品種，種植者也需要其他控制。第27頁，共31頁。This study showed that most of the tested products slowed the progression of bacterial wilt on geranium, but did 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 culture