Evaluation of rhizosphere bacteria and derived bio-organic fertilizers as potential biocontrol agents against bacterial wilt (Ralstonia solanacearum) of potato

Aims

Potato bacterial wilt (Ralstonia solanacearum) is a soil-borne disease that affects the potato plant (Solanum tuberosum) worldwide and causes serious economic losses in southern China. The objective of this study is to study the effect of bacterial antagonists and bio-organic fertilizers on potato bacterial wilt and rhizosphere soil microbial population.

Methods

In the present study, pot and field experiments were conducted to evaluate the LH23 (Bacillus amyloliquefaciens) and LH36 (Bacillus subtilis) strains and their derived bio-organic fertilizers (BIO23 and BIO36) as potential biocontrol agents against potato bacterial wilt.

Results

BIO23 and BIO36 decreased the incidence of bacterial wilt disease and increased potato yields. In pot experiments, the disease incidence of BIO23 and BIO36 was 8.9 % and 11.1 % respectively, much lower than the control (57.7 %). The biocontrol efficiency of BIO23 was 84.6 %, which was the most successful treatment and BIO36 was the second with a biocontrol efficiency of 80.8 %. The increased percentages of potato yields when compared with the control were 63.5 % (BIO23), 64.7 % (BIO36) 34.8 % (LH23), 33.6 % (LH36) and 20.7 % (OF). The counts of antagonists, bacteria and actinobacteria in the rhizosphere soil were significantly increased in BIO23 and BIO36 treatments, whereas the counts of R. solanacearum and fungi in the soil in the both treatments decreased. In field experiments, 70 days after treatment, the biocontrol efficacies of BIO23 and BIO36 treatments were 92.0 % and 84.0 %, and the yield increases of BIO23 and BIO36 treatments were 42.3 % and 28.8 %, respectively, when compared with the organic fertilizer treatment. In addition, the changes in the microbial populations were the same as those observed in the greenhouse experiment.

Conclusions

Potato bacterial wilt could be well controlled by the application bio-organic fertilizer containing a specific antagonist, mainly through the alternation of soil microbial community     

土壤酸化改良剂对烟株农艺性状、土壤pH值及青枯病发生的影响

由于长期连作障碍、不合理连续施用化肥等因素,植烟土壤存在酸化等问题,导致烟草青枯病等愈发严重.土壤酸化改良剂可以改善土壤酸化状况,同时还具有改善土壤物理结构、提高养分利用率、增强土壤酸碱缓冲能力等多项作用.本研究选取3种土壤酸化改良剂,用起垄条施的方式,以长期植烟酸化烟田为研究对象,评估不同处理对土壤pH值调节、烟草生长及青枯病发生的影响.结果表明,草木灰粉剂、草木灰颗粒剂处理对烟株生长有一定的促进作用;草木灰粉剂、草木灰颗粒剂和牡蛎壳粉处理后20d,三者土壤pH值分别较空白对照提高了0.40、0.63和0.40,草木灰颗粒剂对酸化土壤的改良效果最好;处理后60d,三者土壤pH值分别较空白对照提高了0.42、0.30和0.08,草木灰粉剂对酸化土壤的改良效果最好;基于烟草青枯病的病情指数AUDPC计算,从小到大依次为草木灰粉剂、草木灰颗粒剂、牡蛎壳粉、空白对照,草木灰粉剂和草木灰颗粒剂处理对烟草青枯病整个时期的防控效果分别为69.35%和28.43%.结合调酸效果、农艺性状和对青枯病的防效等指标综合分析,土壤酸化改良剂草木灰粉剂起垄条施的效果最优,可为今后在酸化烟田大面积运用调酸技术防控烟草青枯病提供理论基础和技术支撑.     

Rhizocompetence and antagonistic activity towards genetically diverse Ralstonia solanacearum strains – an improved strategy for selecting biocontrol agents

Bacterial wilt caused by Ralstonia solanacearum is a serious threat for agricultural production in China. Eight soil bacterial isolates with activity against R. solanacearum TM15 (biovar 3) were tested in this study for their in vitro activity towards ten genetically diverse R. solanacearum isolates from China. The results indicated that each antagonist showed remarkable differences in its ability to in vitro antagonize the ten different R. solanacearum strains. Strain XY21 (based on 16S rRNA gene sequencing affiliated to Serratia) was selected for further studies based on its in vitro antagonistic activity and its excellent rhizocompetence on tomato plants. Under greenhouse conditions XY21 mediated biocontrol of tomato wilt caused by seven different R. solanacearum strains ranged from 19 to 70 %. The establishment of XY21 and its effects on the bacterial community in the tomato rhizosphere were monitored by denaturing gradient gel electrophoresis of 16S rRNA gene fragments PCR-amplified from total community DNA. A positive correlation of the in vitro antagonistic activities of XY21 and the actual biocontrol efficacies towards seven genetically different R. solanacearum strains was found and further confirmed by the efficacy of XY21 in controlling bacterial wilt under field conditions.     

Silicon-Mediated Tomato Resistance Against Ralstonia solanacearum is Associated with Modification of Soil Microbial Community Structure and Activity

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease of Solanaceae crops. In this study, the soil microbial effects of silicon-induced tomato resistance against R. solanacearum were investigated through pot experiment. The results showed that exogenous 2.0 mM Si treatment reduced the disease index of bacterial wilt by 19.18 % to 52.7 % compared with non-Si-treated plants. The uptake of Si was significantly increased in the Si-treated tomato plants, where the Si content was higher in the roots than that in the shoots. R. solanacearum inoculation resulted in a significant increase of soil urease activity and reduction of soil sucrase activity, but had no effects on soil acid phosphatase activity. Si supply significantly increased soil urease and soil acid phosphatase activity under pathogen-inoculated conditions. Compared with the non-inoculated treatment, R. solanacearum infection significantly reduced the amount of soil bacteria and actinomycetes by 52.5 % and 16.5 %, respectively, but increased the ratio of soil fungi/soil bacteria by 93.6 %. After R. solanacearum inoculation, Si amendments significantly increased the amount of soil bacteria and actinomycetes and reduced soil fungi/soil bacteria ratio by 53.6 %. The results suggested that Si amendment is an effective approach to control R. solanacearum. Moreover, Si-mediated resistance in tomato against R. solanacearum is associated with the changes of soil microorganism amount and soil enzyme activity.     

Control of Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici using leaf extract of Piper betle L.: a preliminary study

The main objective of this study was to evaluate the effectiveness of crude chloroform extract of Piper betle L. (PbC) in controlling Fusarium wilt of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. lycopersici. It was observed that 1% (w/w) amendment of the PbC in soil was more efficient in reducing the Fusarium population in soil than carbendazim and the combined amendment of carbendazim and PbC. Fusarium wilt control studies were carried out in a greenhouse. Variation in different parameters like shoot growth, root growth and mean fresh weights of tomato seedlings in all the treatments were recorded. Accumulation of total phenolics was also studied from the root tissues of tomato. Higher accumulation of total phenolics was observed in the Fusarium-infested plants as compared to that of healthy control and PbC-treated plants. Moreover, it was observed that the extract could reduce the symptoms and disease development. Electron microscopy studies were also done to observe the Fusarium infestation in the vascular bundles and to show the accumulation of total phenolics in the vacuoles of root tissue.     

Effect of combination of bio-agents and mineral nutrients for the management of alfalfa wilt pathogen Fusarium oxysporum f. sp. medicaginis

Abstract

Biological and nutrient management of soil borne disease is increasingly gaining stature as a possible practical and safe approach. Inhibitory effects of fungal and bacterial antagonists were tested under in vitro conditions against the wilt pathogen of alfalfa Fusarium oxysporum f. sp. medicaginis. Trichoderma harzianum and Pseudomonas fluorescens (PI 5) were found to be effective against the alfalfa wilt pathogen. Manganese sulphate at 500 and 750 ppm inhibited the mycelial growth of F. oxysporumf. sp. medicaginis under in vitro conditions. In pot culture studies, manganese sulphate at 12.5 mg/kg reduced the wilt incidence (23.33%). Combined application of manganese sulphate 12.5 mg/kg + T. harzianum 1.25 mg/kg of soil significantly reduced the wilt incidence accompanied by improved plant growth and yield in pot culture. The mixture of manganese sulphate (25 kg/ha) + T. harzianum (2.5 kg/ha) significantly reduced the wilt incidence when applied as a basal dose in the field conditions. The average mean of disease reduction was 62.42% over control.     

家畜粪便堆肥对番茄青枯病、土壤酶活性及土壤微生物功能多样性的影响

合理施用堆肥能够有效地改善植物的生长条件和土壤的生态环境,从而提高植物对病害的抗性。通过盆栽实验,研究了家畜堆肥浸渍液及堆肥混土对番茄青枯病的防治效果及其对土壤酶活性和土壤微生物功能多样性的影响。结果表明,家畜堆肥浸渍液及堆肥混土均对番茄青枯病有一定防治效果,以体积分数1∶1、1∶3的浸渍液处理和质量分数10%的堆肥混土处理效果较好,分别降低病情指数69.4%,31.5%和13.0%。而且浸渍液处理效果优于堆肥混土处理,浓度越高抗病效果越明显。堆肥混土处理可提高土壤脲酶活性,对蔗糖酶和过氧化氢酶活性影响不大;1∶1堆肥浸渍液处理能显著提高土壤脲酶和蔗糖酶活性。基于BIOLOG方法的土壤微生物群落功能研究表明,两种堆肥处理的平均每孔变化率(AWCD)值、Shannon多样性指数、Simpson多样性指数均较对照减小,而对于Alatalo均匀度指数则没有显著影响。不同堆肥处理间微生物碳源利用存在较大差异,堆肥混土处理的主要碳源是糖类和羧酸类物质,而浸渍液处理则是糖类和氨基酸类物质。通过主成分分析得到的堆肥处理聚类结果与各种处理的抗病性强弱分类情况相吻合,且与AWCD值、Shannon多样性指数、Simpson多样性指数的强弱分类也大致吻合。研究表明,施用家畜粪便堆肥主要通过改变土壤微生物群落多样性和土壤酶活性,提高番茄植株的抗病性。     

Isolation and characterization of phosphate solubilizing rhizobacteria to improve plant health of tomato

In the present study, 43 isolates of Phosphate solubilizing rhizobacteria (PSRB) were isolated from 37 rhizospheric soil samples of tomato collected from tomato growing regions of Karnataka. Among the 43 isolates, 33 were found to be positive for solubilizing both inorganic and organic forms of phosphorous. The isolates were analyzed for their ability to colonize roots of tomato and to increase the seed quality parameters under laboratory conditions. On the basis of above criteria, 16 isolates were selected for further studies. Organic acids from PSRB isolates were analyzed and phytase zymogram for two isolates viz., PSRB21 and 31 was prepared. Under greenhouse conditions, all selected isolates showed increased shoot length, root length, fresh weight, dry weight and phosphorous content of tomato seedlings to various extent with respect to control. Analysis of pH and available phosphorous in rhizosphere soil samples of 30 day-old-seedlings revealed that the available phosphorous content was high in rhizospheric soil samples of plants raised from seeds bacterized with PSRB isolates over control. Even though all selected PSRB’s were able to increase the plant growth, only few of them showed protection against fusarium wilt and none of them against early blight.     

抗生素2,4-二乙酰基间苯三酚作为荧光假单胞菌2P24菌株生防功能因子的实证分析

荧光假单胞杆菌2P24菌株分离自小麦全蚀病自然衰退土壤,它是酚类抗生素2,4-二乙酰基间苯三酚(2,4-DAPG)的高产菌,对多种土传病害具有较好的防治能力。利用同源重组构建2,4-DAPG合成基因的定位突变体,并对突变体进行基因互补,通过检测突变菌株和恢复突变菌株抗生素产量和生防效果确定2,4-DAPG在菌株2P24生防功能中的作用。实验中,定位突变体丧失产生抗生素和拮抗病原菌的能力,而恢复突变体的抗生素产量和拮抗能力均恢复至野生菌水平。在对番茄青枯病的防病试验中,2,4-DAPG突变体的防效低且下降快,而恢复突变体的生防能力与野生菌相当,且效果稳定。由此可确定2,4-DAPG是菌株2P24防治番茄青枯病的主要因子,在防效上起关键作用。     

Bioavailability and crop uptake of trace elements in soil columns amended with sewage sludge products

In order to assess the potential impact of long-term sewage sludge application on soil health, the equivalent of about 25 years of agronomic applications of low-metal (`EQ') sewage sludge products were made to greenhouse soil columns. After a 6-year period of `equilibration', during which time successive crops were grown with irrigation by simulated acid rain, the plant-available quantities of trace elements were estimated in the soils by extraction with 0.01 M CaCl₂ at 90 °C, and measured directly by uptake into a crop of red clover (Trifolium pratense L.). Soil pH had a strong influence on the level of extractable and plant-available metals, and because the tested sludge products affected soil pH differently, pH was directly factored into the comparison of different sludge treatments with controls. CaCl₂-extractable levels of several metals (Cu, Zn, Mo), sulfur and phosphorus were found to be higher in the soils amended with organic-rich sludge products than in the control soils. However, extractable Cd and Ni were not significantly elevated by the sludge amendments, presumably because of the low total loading of these metals. Copper, Zn and Mo applied in the form of sludge ash had low soil extractability, suggesting that these trace metals were trapped in high-temperature mineral phases formed during sludge incineration, and resisted subsequent weathering in the soil environment. Extractable soil metals in the alkaline-stabilized sludge treatment were also generally low. Phytotoxicity from the sludge metal loadings (Zn≤125, Cu≤135 kg/ha), was not clearly indicated as long as soil pH was maintained in the 6–7 range by lime amendment. Nevertheless, unexplained depressions in yield were noted with some of the sludge products applied, particularly the dewatered and composted materials. On limed soil columns, the most consistent effect of sludge product amendment on red clover composition was a marked increase in plant Mo.     

利迪链霉菌M01对番茄生长、青枯病发病率及根际细菌群落组成的影响

【背景】利迪链霉菌(Streptomyces lydicus)对多种作物均有较好的促生效果,且对病原真菌具有广谱抑制作用,但该菌对细菌性青枯病的防控研究较少。【目的】探究利迪链霉菌M01能否促进番茄生长并抑制番茄青枯病,以及M01对番茄生长的影响是否通过影响根际细菌群落结构实现。【方法】采用温室盆栽试验和扩增子高通量测序技术研究M01对番茄生长、青枯病发病率及根际细菌群落组成的影响。【结果】施用利迪链霉菌M01的番茄植株鲜重、干重、株高、用土壤与作物分析开发(soil and plant analyzer develotrnent, SPAD)方法测量的叶绿素浓度、根系活力和植株P含量比对照分别提高了22.7%、12.5%、16.0%、28.1%、18.4%和17.9%,其中对株高、SPAD值和植株磷含量影响显著(P<0.05)。M01处理延缓了番茄青枯病的发病时间,接种9周后发病率比对照降低了41.8%。此外,M01对番茄根际细菌群落无显著影响(门水平群落组成,P=0.4;属水平群落组成,P=0.4)。【结论】利迪链霉菌M01可促进番茄植株生长并抑制番茄青枯病,利迪链霉菌M01对番茄生长的影响并非通过调控根际细菌群落实现。     

Mentha spicata and Salvia fruticosa composts as soil amendments in tomato cultivation

Τhe potential use of the aromatic plants Mentha spicata L. (spearmint) and Salvia fruticosa Mill. (sage) as soil amendments was evaluated. For this purpose, tomato seeds were sown in pots that had been filled with composts made from these plants and mixed with soil collected from an organically cultivated tomato field. A 2?×?2?×?4 [two types of fertilizer (synthetic and organic), two types of compost (M. spicata and S. fruticosa) and four compost rates (0%, 2%, 4% and 8%, w/w)] factorial experiment was used; the experiment was conducted twice in a growth chamber and lasted 60 days. At 0, 20, 40 and 60 days, after the establishment of the experiment, the soil bacterial and fungal abundance, the growth of nitrifying bacteria, the number of emerging weeds and the shoot length of tomato plants were measured in all treatments; at the end of the experiment, the above and belowground biomass of tomato plants was also determined. Soil microbial density increased with increasing compost rate of both species; the highest fungal and bacterial densities were recorded at 40 and 60 days, after the establishment of the experiment, respectively. Nitrifying bacteria were present in all treatments and at all sampling times. Both composts had a stimulating effect on tomato growth, which was remarkably pronounced with M. spicata. In contrast, weed emergence was reduced, but only in soils amended with M. spicata. The results suggest that M. spicata compost added at a rate of 4% to 8% is a very promising soil amendment, since it stimulates tomato growth, increases soil bacterial and fungal abundance and inhibits weed emergence. Further research is needed to elucidate the mode of action of M. spicata compost, its effect under field conditions and its possible use in mixed crop, rotational crop or cover crop systems.     

Bacterial wilt of tomato in Karnataka and its management by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Field surveys undertaken in major tomato growing districts of the Karnataka state, located in southern part of India, revealed a high incidence of bacterial wilt caused by Ralstonia solanacearum and it is one of the most destructive bacterial diseases of economically important crops. Across all the tomato cultivars under evaluation, the disease incidence in plants ranged from 9% to 39% whereas the incidence in seeds ranged from 4% to 18%. The effects of tomato seed treatments with Pseudomonas fluorescens in the control of bacterial wilt under greenhouse conditions revealed that the treatments protected plants against soil-borne infections of the bacterial wilt organism. Seed treatment with antagonistic P. fluorescens strain significantly improved the quality of seed germination and seedling vigour. The disease incidence was significantly reduced in plants raised from P. fluorescens treated seeds followed by challenge inoculation with R. solanacearum. Periodic field surveys for the incidence of bacterial wilt of tomato could be recommended to monitor the populations of the bacterial wilt pathogen. Workable measures are presented that could lead to the reduction of the prevalence of this serious disease in affected fields of the small farm-holders.     

Garden-waste-vermicompost leachate alleviates salinity stress in tomato seedlings by mobilizing salt tolerance mechanisms

The incidence of salinity-induced plant stress as a result of natural and anthropogenic factors in arid and semi-arid agricultural lands is great. In South Africa alone, 9 % of irrigated agricultural land is salt-affected. Commercial fertilizers used for improving soil nutrient levels are costly and affect the quality, lifespan and sustainability of soil and water resources. Organic farming practices are based on cost-effective and environmentally-aware management systems. Vermicompost leachate (VCL) is a vermicompost-derived liquid product that has become recognised as a suitable soil amendment product. Commercial tomato (Lycopersicon esculentum Mill var. Heinz-1370) seedlings were subjected to sodium chloride (NaCl) concentrations of 0, 25, 50 and 100 mM and were treated with 1:10 (v/v) WizzardWorms VCL prepared in Hoagland’s nutrient solution under greenhouse conditions. Morphological characters of VCL-treated tomato seedlings showed improved root growth and stimulated overall aboveground growth with significantly higher numbers of leaves, greater stem thickness and increased leaf area, even at a high NaCl-tested concentration (100 mM). The accumulation of compatible solutes such as proline and total soluble sugars indicate an induced salt tolerance or adaptive mechanism in VCL-treated tomato seedlings. The current investigation demonstrates the potential of an organic liquid to maximise tomato productivity by improving seedling growth performance under salt stress conditions.     

Isolation and screening of <Emphasis Type="Italic">phlD</Emphasis><Superscript>+</Superscript> plant growth promoting rhizobacteria antagonistic to <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD ⁺ bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD ⁺ antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92–5.33%), 25 produced indole acetic acid (1.63–7.78 μg ml⁻¹) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD ⁺) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD ⁺ bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD ⁺) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant⁻¹ respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD ⁺ isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.     

Effect of Brassica carinata (L.) biofumigants (seed meal) on chickpea wilt (Fusarium oxysporum f.sp. ciceris), growth,yield and yield component in Ethiopia

Pot experiments were carried out in the green house at Amhara Regional Agriculture Research Institute (ARARI) Bahirdar, Ethiopia to evaluate the potential of Brassica carinata cultivars namely; Holleta-l, S-67 and Yellow Dodola in 2007 and 2008. The treatment effects of B. carinata (L.) cultivars Holleta–1, S-67 and Yellow Dodola seed meals on chickpea fusarium wilt (Fusarium oxysporum f.sp. ciceris) were studied. Six rates of seed (0, 5, 10, 15, 20 and 25 g/kg of infested soil) were used. Infested soil without B.carinata cultivars amendments as a control and susceptible check variety JG-62 also without amendments were used in all the experiments. For each seed meal experiment, the treatments were arranged in factorial randomised complete block design in three replications. Data on seedling emergence, wilt incidence, fresh weight, dry weight, pod per plant, seed per pod, hundred seed weight and yield per hectare were collected. The amendments of infested soil with B.carinata cultivars seed meal reduced the incidence of chickpea fusarium wilt and increased yield per hectare. The interaction of the seed meal Holleta-1, S-67 and Yellow Dodola at 10–25 g/kg infested soil were effective in reducing wilt incidences on chickpea. However, the interaction of Yellow Dodola with 20 and 25 g seed meal per kg infested soil were the best combination in reducing significantly wilt incidence. The three cultivars incorporated at different level of doses significantly affected the influence of Fusarium wilt on the fresh weight, dry weight, pod per plant, seed per pod, hundred seed weight and yield per hectare. The highest yield kg/ha was recorded in combination of Yellow Dodola seed meal at 20 and 25 g followed by S-67 and Holleta-1 at 25 g /kg infested soil, respectively. The interaction of Holleta-1 at 5–25 infested soil significantly reduced disease incidence up to 16.7–43.3% and increased yield per hectare with mean by (30%) over the control. Seed meal amendment S-67 significantly reduce disease incidence 26.7–46.7% and increased yield kg/ha with mean by (36.7%) from the unamended control. Yellow dodola reduces disease incidence with 26.7–63.3% and increased yield kg/ha with mean by (45%) from the unamended control. The result indicates the potential of using Brassica crop seed meal amendment as useful component of integrated chickpea wilt management.     

Comparison of bacterial community structures in the rhizoplane of tomato plants grown in soils suppressive and conducive towards bacterial wilt.

It has been reported that the growth of Ralstonia solanacearum is suppressed at the rhizoplane of tomato plants and that tomato bacterial wilt is suppressed in plants grown in a soil (Mutsumi) in Japan. To evaluate the biological factors contributing to the suppressiveness of the soil in three treated Mutsumi soils (chloroform fumigated soil; autoclaved soil mixed with intact Mutsumi soil; and autoclaved soil mixed with intact, wilt-conducive Yamadai soil) infested with R. solanacearum, we bioassayed soil samples for tomato bacterial wilt. Chloroform fumigation increased the extent of wilt disease. More of the tomato plant samples wilted when mixed with Yamadai soil than when mixed with Mutsumi soil. Consequently, the results indicate that the naturally existing population of microorganisms in Mutsumi soil was significantly able to reduce the severity of bacterial wilt of tomato plants. To characterize the types of bacteria present at the rhizoplane, we isolated rhizoplane bacteria and classified them into 22 groups by comparing their 16S restriction fragment length polymorphism patterns. In Yamadai soil a single group of bacteria was extremely predominant (73.1%), whereas in Mutsumi soil the distribution of the bacterial groups was much more even. The 16S rDNA sequence analysis of strains of dominant groups suggested that gram-negative bacteria close to the beta-proteobacteria were most common at the rhizoplane of the tomato plants. During in vitro assays, rhizoplane bacteria in Mutsumi soil grew more vigorously on pectin, one of the main root exudates of tomato, compared with those in Yamadai soil. Our results imply that it is difficult for the pathogen to dominate in a diversified rhizobacterial community that thrives on pectin.     

Bioorganic Fertilizer Enhances Soil Suppressive Capacity against Bacterial Wilt of Tomato

Tomato bacterial wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases. Many strategies have been taken to improve soil suppressiveness against this destructive disease, but limited success has been achieved. In this study, a novel bioorganic fertilizer revealed a higher suppressive ability against bacterial wilt compared with several soil management methods in the field over four growing seasons from March 2011 to July 2013. The application of the bioorganic fertilizer significantly (P<0.05) reduced disease incidence of tomato and increased fruit yields in four independent trials. The association among the level of disease incidence, soil physicochemical and biological properties was investigated. The soil treated with the bioorganic fertilizer increased soil pH value, electric conductivity, organic carbon, NH₄ ⁺-N, NO₃ ^--N and available K content, microbial activities and microbial biomass carbon content, which were positively related with soil suppressiveness. Bacterial and actinomycete populations assessed using classical plate counts were highest, whereas R. solanacearum and fungal populations were lowest in soil applied with the bioorganic fertilizer. Microbial community diversity and richness were assessed using denaturing gel gradient electrophoresis profile analysis. The soil treated with the bioorganic fertilizer exhibited higher bacterial community diversity but lower fungal community diversity. Redundancy analysis showed that bacterial community diversity and richness negatively related with bacterial wilt suppressiveness, while fungal community richness positively correlated with R. solanacearum population. We concluded that the alteration of soil physicochemical and biological properties in soil treated with the bioorganic fertilizer induced the soil suppressiveness against tomato bacterial wilt.     

Isolation and characterization of Pseudomonas brassicacearum J12 as an antagonist against Ralstonia solanacearum and identification of its antimicrobial components

A bacterial strain, J12, isolated from the rhizosphere soil of tomato plants strongly inhibited the growth of phytopathogenic bacteria Ralstonia solanacearum. Strain J12 was identified as Pseudomonas brassicacearum based on its 16S rRNA gene sequence. J12 could produce 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide (HCN), siderophore(s) and protease. The maximum growth and antagonistic activity were recorded at 30°C and pH 8. Glucose and tryptone were used as the most suitable carbon and nitrogen sources, respectively. Strain J12 significantly suppressed tomato bacteria wilt by 45.5% in the greenhouse experiment. The main antimicrobial compound of J12 was identified as 2,4-diacetylphloroglucinol (2,4-DAPG) by HPLC-ESI-MS analysis. The gene cluster phlACBD, which is responsible for 2,4-DAPG production, was identified and expressed in the bacterial strain Escherichia coli DH5α.     

The Relation of Different Crop Roots Exudates to the Survival and Suppressive Effect of Stenotrophomonas maltophilia (PD4560), Biocontrol Agent of Bacterial Wilt of Potato

Experiments were performed under greenhouse conditions to control bacterial wilt of potato (potato brown rot), caused by Ralstonia solanacearum race 3 biovar 2, Phylotype II, sequevar 1 using various biocontrol strategies. These strategies involved the use of the bacterial biocontrol agent Stenotrophomonas maltophilia (PD4560), in clay or sandy soils, planted with cowpea, maize or tomato which was grown separately in different pots in the inoculated soils. After harvest, the soil derived from each cultivated crop was inoculated with a mixture of three virulent R. solanacearum strains (K3, K10 and K16) to achieve a final concentration of 5 × 10⁸ cfu/g dry soil and used in pots under greenhouse conditions to cultivate potato seed tubers. The highest survival of S. maltophilia in soil (more than 160 days) coincided with a remarkable suppressing effect on disease incidence caused by R. solanacearum that expressed by wilt severity (up to 100% reduction), area under disease progress curve (AUDPC) (up to 99% reduction) and counts of the pathogen in soil (up to 75% reduction), rhizosphere (up to 80% reduction) and plant tissue (up to 97% reduction) of potato plants. The amino acid analysis of root exudates of crops under investigation revealed high percentages of asparagines (15.5–21%), glutamine (16–20%) and sulphur‐containing methionine (7–9%) in both of the cowpea and maize, respectively. In tomato root exudates, high percentages of arginine (around 26%) and lysine (around 23%) were detected. Methionine is known to favour the growth of S. maltophilia suggesting that especially cowpea and maize are suitable for crop rotation with potato and will enhance the sustainability of the biocontrol agent S. maltophilia.