Control of soil rot of sweet potato by the incorporation of the fresh aerial tissue of Geranium carolinianum L. into the soil

In a previous study, we found that a 70% aqueous ethanol extract of the fresh aerial tissue of Geranium carolinianum L. showed antimicrobial activity against the pathogen causing soil rot of sweet potato. As the appropriate time for cultivation of sweet potato and the growing period of G. carolinianum do not overlap in Okinawa Prefecture, Japan, the fresh aerial tissue is available in order to control soil rot of sweet potato. Thus, we examined the control effect of fresh aerial tissue against soil rot of sweet potato. The various trials (a single repetition of 20 m²) were performed in fields that had undergone 8 years of continuous cropping of sweet potato at Okinawa Prefectural Agricultural Experiment Station, Horticultural branch. After harvest, when the disease severity was evaluated by determining the necrotic area of the storage root, the incorporation of fresh aerial tissue (5000 kg 1000 m^-2) into the soil was considered to be highly effective, with a protective value of 75.4. This result shows that G. carolinianum could be used as a biological agent for the control of soil rot of sweet potato.     

Identification and activity of ethyl gallate as an antimicrobial compound produced by Geranium carolinianum

We isolated an antimicrobial compound from the aerial tissue of Geranium carolinianum and identified it as ethyl 3, 4, 5-trihydroxy benzoate (ethyl gallate) by ¹H-NMR and ¹³C-NMR and gas chromatography–mass spectrometry. The antimicrobial activity of ethyl gallate against three potato pathogens was assayed by the paper disk method. The activity against Ralstonia solanacerum , Streptomyces scabies , and Streptomyces acidiscabies was observed at concentrations >200, >300, and >300 µg disk⁻¹, respectively. These results suggest that the antimicrobial activity of Geranium carolinianum against soil-borne plant disease pathogens is partly related to ethyl gallate.     

青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析

 利用青枯雷尔氏菌(Ralstonia solanacearum)无致病力菌株防治番茄青枯病具有很好的应用潜力。作者通过分离筛选自然弱毒株、⁶⁰Co辐射诱变和EZ-Tn5插入诱变,分别获得3、12和40株青枯雷尔氏菌无致病力突变菌株。经盆栽番茄苗致病性检测,15 d后均未发病,证实均为无致病力青枯雷尔氏菌。进一步对番茄青枯病的防治试验表明,从番茄青枯病发病田块分离的无致病力突变菌株FJAT1458的防治效果最好,防效达100%。该菌株能定殖番茄植株根系土壤、根部和茎部,定殖数量均表现为“先增后减”的趋势,并且接种浓度越大、苗龄越小,定殖数量越大。从构建的防效模型可以看出,不同接种浓度条件下,植株发病率随时间变化符合的回归方程不同,相关系数R值也不同,接种浓度越大,R值越小。本研究获得的青枯雷尔氏菌无致病力突变菌株FJAT1458对番茄青枯病具有很好的防病效果。     

番茄枯萎病和青枯病拮抗细菌的筛选、评价与鉴定

从宁夏银川、江苏沭阳和福建厦门的番茄、辣椒、西瓜等作物根际土壤中,分离纯化获得367株细菌菌株。以番茄枯萎病菌Fusarium oxysporum f.sp.lycopersici和番茄青枯病菌Ralstonia solanacearum为靶标菌,从367株菌株中筛选出对两种病菌皆具有很强拮抗作用的菌株22株。拮抗细菌抑菌物质的研究结果表明：22株拮抗细菌均能分泌蛋白酶;不能分泌几丁质酶;3株细菌能分泌纤维素酶;3株细菌能分泌嗜铁素。盆栽试验结果表明：拮抗细菌PTS-394对番茄枯萎病和青枯病的防效最高,分别为77.4%和80%;菌株H-70、L-1和SJ-280对番茄枯萎病和青枯病的防效均大于60%。对上述4株拮抗细菌进行16S rRNA种属鉴定,均为枯草芽孢杆菌Bacillus subtilis。     

抗不同生化型青枯菌的生防菌筛选鉴定及其活性分析

为更好地利用生防菌控制青枯病危害,从不同地区的土壤中分离到569株细菌菌株,筛选到3株对5种不同生化型青枯劳尔氏菌Ralstonia solanacearum具有较强拮抗活性的菌株,其中菌株BS2004的拮抗活性最强。以BS2004的菌悬液为对照,分别测定无菌滤液、蛋白酶K及高温热处理后拮抗物质抑菌活性的变化。结果显示,蛋白酶K及高温热处理后,该菌的抑菌活性显著降低,表明其主要抑菌成分为蛋白类物质。在设施栽培条件下用生防菌BS2004菌悬液处理番茄植株,能有效控制番茄青枯病的发生,防治效果达66.75%,同时还发现,重新分离得到的青枯菌菌体数明显受到生防菌的抑制。通过对BS2004的形态、生理生化特征、脂肪酸鉴定、16S rDNA序列等进行分析,该菌株被鉴定为解淀粉芽孢杆菌Bacillus amyloliquefaciens。     

两株植物根际促生菌对番茄青枯病的生物防治效果评价

研究了Erwinia persicinus RA2和Bacillus pumilus WP8浸种和拌土处理对番茄青枯病的实际防治效果,及其对番茄根际微生物群落的影响。结果显示,两株菌都具有防治番茄青枯病的作用,并能不同程度地促进番茄幼苗生长。主要体现在显著提高番茄幼苗健株率,病原菌处理的健株率最低,仅为22.4%,而RA2和WP8浸种处理分别达68.9%和62.8%;促进幼苗地上部增高、增粗和根部生长,如WP8浸种处理的茎叶干重和根干重分别达到4.87 mg·株^-1和35.69 mg·株^-1,分别比病原菌对照提高110.82%和205.83%。浸种处理的促进效应明显优于拌土处理;还能在一定程度上提高土壤水稳性团聚体（〉0.25 mm）比例,WP8浸种处理尤为明显,分别比空白对照和病原菌对照提高269.91%和156.88%。DGGE指纹图谱表明根际微生物群落受番茄种植的影响最大,其次是青枯病菌,而受这两种菌施用的影响最小。     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.     

Effects of intercropping and soil amendment with urea and calcium oxide on the incidence of bacterial wilt of tomato and survival of soil-borne Pseudomonas solanacearum in Taiwan

Intercropping and soil amendment experiments were conducted to determine if they reduced populations of Pseudomonas solanacearum and bacterial wilt of tomato at the Asian Vegetable Research and Development Center (AVRDC) and at three other locations in Taiwan. At AVRDC, intercropping tomato with cowpea planted within the row significantly reduced bacterial wilt ( P  < 0.05) compared to when tomato was cropped alone. The P. solanacearum population in soil was not affected by intercropping with cowpea, soybean, or Welsh onion. At the same site, however, a preplanting soil amendment consisting of urea (200 kg ha⁻¹ N) and CaO (5000 kg ha⁻¹) significantly reduced the pathogen population and tomato bacterial wilt ( P  < 0.001). The effect of the soil amendment was not consistent when applied to soil from three other sites in Taiwan; in soil from two sites no reduction of the pathogen population occurred. At these sites, tomato bacterial wilt in the field was not reduced significantly after amending. In comparison with a non-amended control, the addition of only CaO reduced the P. solanacearum population in AVRDC soil significantly ( P  < 0.05), but the reduction was significantly greater when the complete soil amendment was added. In contrast, urea alone did not affect the survival of P. solanacearum in the soil. In a greenhouse experiment with AVRDC soil, P. solanacearum was undetectable 2 weeks after soil amendment, but in the same treatment tomato yield was significantly reduced by 48% ( P  < 0.05) compared with non-amended treatments. The suppressive effect of the soil amendment on the P. solanacearum population was probably due to the generation of one or several toxic substances during the transformation of urea in the presence of CaO.     

马铃薯青枯病的PCR检测

以我国11个省(市、区)和6种不同寄主植物的43个青枯菌Ralstonia solanacearum代表性菌株和4个国外青枯菌菌株为试材,采用15条随机引物进行了RAPD分析,筛选到了1条青枯菌所特有的DNA片段,根据其碱基序列,设计了特异PCR引物,对不同青枯菌DNA进行扩增,并以马铃薯的其它病原细菌为对照,只有青枯菌可获得773bp的DNA扩增产物.经过对PCR反应模板制备程序的简化和优化,利用本研究设计的特异引物,直接对马铃薯病块茎的DNA粗提液进行扩增,获得了773bp片段.此技术可望用于马铃薯种薯青枯病菌的检测,大大缩短检测时间,提高检测效率.     

烟草根际细菌铜绿假单胞菌swu31-2的定殖能力及其对烟草青枯病的防治作用

从重庆黔江烟草田间分离获得一株烟草青枯菌拮抗菌株Pseudomonas aeruginosa swu31 2（简称swu31 2）。采用逐步提高药物浓度的方法,筛选获得了抗链霉素300 μg/mL对烟草青枯病菌拮抗活性稳定的swu31 2突变菌株。采用灌根接种法,研究其在烟草根、茎和叶表面及内部的定殖能力及其对烟草青枯病的防治作用。结果表明,swu31 2能在烟草各组织的表面及内部定殖。该菌株在烟草各组织内部的数量均表现为“由增到减”的趋势。在接种后第8天定殖数量达到最高峰,随后有所下降;到第20天各组织内的数量仍然维持较高水平（105 cfu/g 以上）。同时,在接种20 d后,烟草的根、茎和叶的表面仍然可以检测到swu31 2的存在。盆栽试验结果表明,swu31 2的菌液和活性物质对烟草青枯病均有一定的防治效果。其中先施swu31 2菌液和活性物质粗提物的防效好于农用链霉素（51.25%）,分别为60.87%和 60.32%,而后施菌液和活性物质粗提物的防效也分别达39.50%和20.90%。     

娄彻氏链霉菌XL-6的抑菌活性及对茄子幼苗的防病促生效应

本文研究了娄彻氏链霉菌Streptomyces rochei XL-6无菌发酵液在自然状态下的抑菌活性及对茄子幼苗的防病促生效果。结果表明,菌株XL-6发酵液的抑菌活性物质可耐70℃高温,在pH 5~8其抑菌活性保持稳定;该抑菌活性物质不受紫外线、蛋白酶K和胰蛋白酶的影响,在4℃条件下贮藏28 d仍可保持良好的抑菌活性。采用不同浓度的发酵液处理茄子幼苗均可提高茄苗对青枯病的抗性,其中,施用40倍发酵液显著降低茄子青枯病发病率和病情指数并显著提高PAL和PPO酶活性,对盆栽茄子的防治效果达64.78%。同时,40倍发酵液显著提高茄子种子发芽率、鲜重、叶绿素含量及根系活力,较病原菌菌体处理和无菌水处理,茄苗干重增幅分别达15.81%和27.95%,对茄苗的促生效果显著。说明适宜浓度的拮抗菌XL-6发酵液兼具防治茄子青枯病和促进植株生长的效果。     

青枯病和根肿病生防细菌BS2004的发酵培养基和培养条件优化

解淀粉芽孢杆菌BS2004是一株对不同生化型的番茄青枯病菌Ralstonia solanacearum和十字花科根肿病菌Plasmodiophora brassicae具有抑制作用的优良生防菌,主要依靠产生拮抗物质抑制病菌.为提高其抑菌活性物质的产量与防治效果,本文采用正交试验和单因素试验设计,通过摇瓶培养对其发酵培养基和培养条件进行了优化.优化后的发酵培养基配方为蛋白胨 10 g·L^-1、牛肉浸膏 1 g·L^-1、酵母粉 1 g·L^-1和Nacl 8 g·L^-1;优化后的培养条件为pH 7.5、温度 35 ℃、装瓶量 35 mL·50 mL^-1、接种量 3%（v/v）和最佳培养时间 24 h.经条件优化后培养的 BS2004对青枯病菌的抑菌圈直径达 21.75 mm,对番茄青枯病的防治效果达 80%,对十字花科根肿病的防治效果达 60%,增产 1.92 倍.     

Behavior of Ralstonia solanacearum Race 3 Biovar 2 During Latent and Active Infection of Geranium

ABSTRACT Southern wilt of geraniums (Pelargonium hortorum), caused by the soilborne bacterium Ralstonia solanacearum race 3 biovar 2 (R3bv2), has inflicted significant economic losses when geranium cuttings latently infected with this quarantine pest were imported into the United States. Little is known about the interaction between R. solanacearum and this ornamental host. Using UW551, a virulent R3bv2 geranium isolate from a Kenyan geranium, we characterized development of Southern wilt disease and R3bv2 latent infection on geranium plants. Following soil inoculation, between 12 and 26% of plants became latently infected, carrying average bacterial populations of 4.8 x 10(8) CFU/g of crown tissue in the absence of visible symptoms. Such latently infected plants shed an average of 1.3 x 105 CFU/ml in soil run-off water, suggesting a non-destructive means of testing pools of asymptomatic plants. Similarly, symptomatic plants shed 2 x 10(6) CFU/ml of run-off water. A few hundred R. solanacearum cells introduced directly into geranium stems resulted in death of almost all inoculated plants. However, no disease transmission was detected after contact between wounded leaves. Increasing temperatures to 28 degrees C for 2 weeks did not convert all latently infected plants to active disease, although disease development was temperature dependent. Holding plants at 4 degrees C for 48 h, a routine practice during geranium cutting shipment, did not increase frequency of latent infections. R. solanacearum cells were distributed unevenly in the stems and leaves of both symptomatic and latently infected plants, meaning that random leaf sampling is an unreliable testing method. UW551 also caused potato brown rot and bacterial wilt of tomato, surpassing race 1 strain K60 in virulence on tomato at the relatively cool temperature of 24 degrees C.     

2株分泌型铁载体真菌对番茄青枯病的防效

为筛选对番茄青枯病防治效果好的天然产物,本文以分泌型铁载体产生菌云南木霉Trichoderma yunnanense 2-14F2和拟球孢白僵菌Beauveria pseudobassiana 2-8F2为材料,考察其铁载体活性物质对番茄青枯病的防效。采用CAS检测法及全波段紫外光吸收法判定铁载体化学结构类型及其活性。采用Sephadex-LH20凝胶划段法分离活性物质。采用平板扩散及96孔板倍半稀释法检测铁载体活性物质对青枯雷尔氏菌Ralstonia solanacearum的离体抑菌活性;采用盆栽法检测活性物质对番茄青枯病的防效。结果显示,菌株2-14F2和2-8F2产铁载体活性单位(SU)分别为62.02%和52.06%,铁载体化学结构类型均为异羟肟酸盐型(hydroxamates)。当铁载体活性物质浓度为0.15 mg/mL时,两菌株对青枯雷尔氏菌抑菌率分别为73.26%(2-14F2)和37.23%(2-8F2);对番茄青枯病的防治效果分别达到51.36%(2-14F2)与50.27%(2-8F2)。此外,当两菌株铁载体活性物质中含1 mol/L FeCl₃     

茄科作物青枯病研究进展

 茄科细菌性青枯病是世界性的严重性病害,具有寄主范围广,防治困难的特点,一直是国内外研究的焦点和热点。本文结合最新的研究内容,就该病病原菌的分类情况、致病机制、全基因组测序、检测方法和防治措施等进行了概述。     

马铃薯枯萎病生防芽胞杆菌筛选及生防效果研究

马铃薯枯萎病是马铃薯主要土传真菌病害之一，已成为限制我国马铃薯产业健康发展的重要因素之一。菌株ZF128是本实验室从马铃薯根际土壤分离筛选得到的一株对马铃薯枯萎病菌具有显著抑制效果的生防细菌。经生理生化、BIOLOG GENIII和系统发育树分析，鉴定菌株ZF128为贝莱斯芽胞杆菌Bacillus velezensis。抑菌谱测定结果显示，ZF128对5种病原真菌和5种病原细菌都具有显著拮抗效果。光学显微观察发现，菌株ZF128处理的病原菌菌丝生长受到抑制并出现膨胀变粗现象。发酵试验表明，菌株ZF128在甘露醇培养基中发酵上清液对马铃薯枯萎病菌的抑制效果最好。盆栽试验结果证明，接种菌株ZF128后马铃薯发病明显减轻，防效为82.46%。此外，定殖和促生试验表明，菌株ZF128能在马铃薯根际和根部有效定殖30d以上，处理组马铃薯株高、根长、地上部和地下部鲜重及叶绿素含量等指标均显著高于对照组。综上，菌株ZF128生防性状优良，是一株具有潜在应用价值的芽胞杆菌。     

无致病力青枯菌株对番茄青枯病的防治效果

用紫外诱变法获得的青枯无致病力菌株ATm0 4 4和Asp0 6 1对致病菌没有直接的抑制作用 ;处理番茄后对青枯病产生抗性 ;两菌株可在番茄体内定殖并繁殖 ,移栽浸根是最佳的处理方法。盆栽试验结果表明 ,番茄经ATm0 4 4和Asp0 6 1处理后 ,分别较对照推迟 9和 7d发病 ,2 0d后的防效达 56 .7%和 53.4 %。田间小区试验结果表明 ,经ATm0 4 4和Asp0 6 1菌悬液浸根处理番茄 ,1 5d后对青枯病的防治效果分别为 53.8%和 37.7%。     

Rapid field detection of Ralstonia solanacearum in infected tomato and potato plants using the Staphylococcus aureus slide agglutination test

Ralstonia solanacearum is the major cause of bacterial wilt, which affects over 200 species of plants, many of economic importance. Current methods for detection and identification of the pathogen rely on isolation on semi-selective media followed by a combination of serological and molecular techniques and plant inoculation. Such methods are time-consuming, and require extensive laboratory facilities and skilled personnel. A reliable and rapid screening technique, which could be applied in the field, would reduce the number of samples submitted for laboratory testing and provide a vital component in disease control measures. A programme for the control of R. solanacearum has been introduced in Portugal and a Staphylococcus aureus slide agglutination test was used directly on tomato and potato plants in the field and on bacterial cultures under laboratory conditions. The results obtained show that this technique can play a major role in control programmes by providing a sensitive tool for the rapid detection of the pathogen.     

抑制植物病原细菌的植物筛选

以3种重要的植物病原细菌—辣椒青枯病菌(Ralstonia solanacearum)、柑橘溃疡病菌(Xanthomonas citri)和大白菜软腐病菌(Erwinia carotovorapv.carotovora)为供试菌,对43种植物甲醇提取物进行离体抑菌活性测定。研究结果表明,漆树对辣椒青枯病菌抑菌活性最强,其次是金秀清明茶;抑制柑橘溃疡病菌活性最高的植物有漆树和十大功劳;对大白菜软腐病菌没有筛选到抑菌活性较好的植物;垫状卷柏、七叶一枝花、少花龙葵、水半夏和豆瓣菜粗提物对3种植物病原细菌均无抑制作用。     

Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods are described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R. solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74–97, 455–475, 1454–1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division II (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii , or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum , 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.