Seedling Petri-dish inoculation method:A robust,easy-to-use and reliable assay for studying plant-Ralstonia solanacearum interactions

Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops,leading to huge losses in crop production every year.Understanding of plant-R.solanacearum interactions will aid to develop efficient strategies to control the disease.As a soilborne pathogen,R.solanacearum naturally infects plants via roots.A huge limitation in studying plant-R.solanacearum interactions is the large variation of R.solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.Here,we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.This method is easy to use,takes about only 10 days from seed germination to the completion of inoculation assay,and requires less inoculum of bacteria as well as growth chamber space.We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns,resistance of defense-related plant mutants,and virulence of R.solanacearum mutants.Furthermore,we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

Microstructure characteristics and solidification behavior of wrought aluminum alloy 2024 rheo-diecast with self-inoculation method

One important problem in casting wrought aluminum alloys is the high tendency to the formation of hot tears in the solidification process.By using semi-solid metal(SSM) processing,the hot tearing tendency of alloys can be minimized during casting.In the present research,the semi-solid slurry of wrought aluminum alloy 2024 was firstly prepared with a novel self-inoculation method(SIM),and then the microstructure characteristics of the semi-solid slurry and the rheo-diecastings cast with the semi-solid slurry were investigated.The results indicate that finer and more uniform globular primary α-Al particles can be obtained when the semi-solid slurry are isothermally held for a short period within the semi-solid temperature range,and the primary α-Al particles without entrapped liquid are uniformly fine,globular grains in the rheo-diecastings.The holding temperature and time affect the solid fraction,particle size,and shape factor.After the semi-solid slurry is held at 625 ℃ for 3 min and 5 min,the optimal values for the average equivalent diameter are 70.80 μm and 74.15 μm,and for the shape factor are 1.32 and 1.42,respectively.The solidification process of the rheo-diecastings is composed of the following two distinct stages:primary solidification process and secondary solidification process.The secondary solidification process consists further of the following three stages:(1) direct growth of secondary primary(α 2) phase from the surface of the primary α-Al phase particles without re-nucleation,(2) independent nucleation and growth of α 3 phase from the residual liquid,and(3) eutectic reaction at the end.

Microstructure of partially remelted billet of AM60 alloy prepared with self-inoculation method

The billets of AM60 alloy, prepared with self-inoculation method, were partially remelted into semisolid state.Effects of process parameters on remelting microstructure of semisolid billet were investigated.Experimental results show that the solid particles obtained with self-inoculation method are in smaller grain size and globular shape after partial remelting, compared with those prepared with other casting methods.In the optimized process conditions, the average size of solid particles of partially remelted billet is 65 μm, and the shape factor is 1.12.The process parameters, i.e.pouring temperature, addition amount of self-inoculants, and the slope angle of multi-stream mixing cooling channel have influence on the microstructure of partially remelted billet.The optimized temperature is from 680 °C to 700 °C, addition amount of self-inoculants is between 5% and 7% (mass fraction), slope angle of multi-stream mixing cooling channel is between 30° and 45°, with which the dendritic microstructure of as-cast billet can be avoided, and the size of solid particles of remelted billet is reduced.

Effects of novel self-inoculation method on microstructure of AM60 alloy

A novel cast processing method,self-inoculation method (SIM),was proposed.The process involves the addition of self-inoculant to melt,then pouring the melt to a mould through a multi-stream mixing cooling channel.In this paper,the process parameters were investigated.Results indicate that the melt treatment temperature,the amount of self-inoculant added,and the slope angle of the cooling channel are the key factors for SIM process.The optimized parameters are that the melt treatment temperature is between 680 and 700°C;the addition of self-inoculant is between 5wt.% and 7wt.%;and the slope angle of the cooling channel is between 30° and 45°.Further analysis reveals that SIM changes the solidification microstructure of slurry by controlling the nucleation and growth of the primary phase in the melt.

Solidification behavior and rheo-diecasting microstructure of A356 aluminum alloy prepared by self-inoculation method

Semisolid slurry of A356 aluminum alloy was prepared by self-inoculation method, and the microstructure and solidification behavior during rheo-diecasting process were investigated. The results indicate that the semisolid slurry of A356 aluminum alloy can be prepared by self-inoculation method at 600 °C. Primary α-Al particles with fine and spherical morphologies are uniformly distributed when the isothermal holding time of slurry is 3 min. Liquid phase segregation occurs during rheo-diecasting process of semisolid slurry and the primary particles(α1) show obvious plastic deformation in the area of high stress and low cooling rate. A small amount of dendrites resulting from the relatively low temperature of the shot chamber at the initial stage of secondary solidification are fragmented as they pass through the in-gate during the mould filling process. The amount of dendrite fragments decreases with the increase of filling distance. During the solidification process of the remaining liquid, the nucleation rate of secondary particles(α2) increases with the increase of cooling rate, and the content of Si in secondary particles(α2) are larger than primary particles(α1). With the increase of cooling rate, the content of Si in secondary particles(α2) gradually increases. The morphologies of eutectic Si in different parts of die casting are noticeably different. The low cooling rate in the first filling positions leads to coarse eutectic structures, while the high cooling rate in the post filling positions promotes small and compact eutectic structures.

Influence of different inoculation methods on the evaluation of the resistance to rice sheath blight(Rhizoctonia solani Kuhn)

To establish a standard system for geneticstudies on sheath blight resistance, a field testwas conducted at the experimental farm ofYangzhou University to compare several pro-cedures for inoculating rice plants with R.solani Kuhn (RH9). The varieties used wereJasmine 85, Teqing (resistant or moderatelyresistant), and Lemont (susceptible). They

Effect of Different Inoculation Methods on Pathogenicity of Fusarium proliferatum to Alfalfa

[Objectives]To find out the suitable inoculation method for Fusarium proliferatum.[Methods]Longmu 801 and TG4 were inoculated with rice grain-based inoculum at the bud and seedling stages and with root irrigation at the seedling stage,respectively,and then,the plant height,aboveground biomass,underground biomass,incidence rate and disease index of the alfalfa were determined to conduct comprehensive resistance evaluation.[Results]There was obvious difference in disease resistance between the two alfalfa varieties.Among the three inoculation methods,the relative plant height,relative aboveground biomass and relative underground biomass of Longmu 801 differed insignificantly(P>0.05),all around 100.There were significant differences in TG4 among different inoculation methods(P<0.05).The incidence rates and disease indices of Longmu 801 and TG4 inoculated with rice grain-based inoculum at the bud stage were the highest,and their incidence rates and disease indices were 73.3%and 100%,and 40 and 60,respectively,significantly higher than those under the other two inoculation methods.The incidence rates and disease indices of Longmu 801 and TG4 inoculated with rice grain-based inoculum at the seedling stage were the lowest,and their incidence rates and disease indices were 0%and 13.30%,and 0 and 5.33,respectively,significantly lower than those under the other two inoculation methods.[Conclusions]The inoculation time and inoculation method of the pathogen both affect the inoculation effect.Among the three inoculation methods,the inoculation with rice grain-based inoculum at the bud stage is the most pathogenic to alfalfa seedlings,and can be popularized in the resistance evaluation of alfalfa to root diseases.

In-vitro vs in-vivo Inoculation: Screening for Resistance of Australian Rice Genotypes Against Blast Fungus

To assist with rapid screening for rice blast resistance as a precursor in a breeding program, the susceptibility to rice blast of 13 rice genotypes from Australia was evaluated in May to June 2013 using three distinct inoculation methods（spot, filter paper and standard methods） at seedling, vegetative and reproductive stages. The results revealed that the spot and filter paper inoculation methods were successful in discerning susceptibility to the rice blast disease（P ≤ 0.05）. Disease susceptibility declined significantly from the vegetative to reproductive stages. The standard method was conducted at three different stages for pot plants grown inside the mist house. However, low temperatures did not produce disease symptoms except in a few genotypes. Among the 13 rice genotypes screened, AAT9 expressed a highly resistant response, and AAT4, AAT6, AAT10, AAT11, AAT13, AAT17 and AAT18 expressed resistance at various stages. The results will be useful for selecting elite genotypes for disease tolerance where rice blast is prevalent. In addition, the resistant genotypes can serve as a gene pool used in breeding programmes to develop new resistant genotypes.

Establishment and Application of Identification Methods for Resistance to Sugarcane White Leaf Disease(SCWL)

[Objectives]This study was conducted to establish simple, efficient, stable, standardized and practical identification methods for sugarcane resistance to white leaf disease(SCWL), and promote the breeding for sugarcane resistance to SCWL. [Methods]The identification technology of sugarcane resistance to SCWL was systematically studied and explored from the aspects of sugarcane material treatment and planting, inoculation liquid preparation, inoculation method, disease investigation, grading standard formulation, etc., and two sets of simple, efficient, stable, standardized and practical accurate identification methods for sugarcane resistance to SCWL were created for the first time, namely, the seed cane coating inoculation method and the stem-cutting inoculation method at the growth stage. The seed cane coating inoculation method includes the steps of directly screening SCWL phytoplasma, extracting juice from cane and adding 10 times of sterile water to prepare an inoculation liquid, spraying seed cane on plastic film to keep moisture, planting the inoculated materials in barrels in an insect-proof greenhouse for cultivation, investigating the incidence rate 30 d after inoculation, and evaluating the disease resistance according to the 1-5 level standard. The method of stem-cutting inoculation includes the steps of directly screening sugarcane stems carrying SCWL phytoplasma and adding 10 times of sterile water to prepare an inoculation liquid, cultivating the identification materials in an insect-proof greenhouse, dropping 100 μl of the inoculation liquid into each root incision with a pipette gun at the age of 6 months, investigating the incidence rate 20 d after planting, and evaluating the disease resistance according to the 1-5 level standard. [Results] The two methods are similar to the natural transmission method. After inoculation, SCML occurred significantly, with high sensitivity and good reproducibility. The results of resistance identification were consistent with those of natural disease in the field. Through the two inoculation methods and field natural disease investigation, the resistance of 10 main cultivars to SCML was identified, which was true and reliable. [Conclusions] This study can provide standard varieties for identification of SCML resistance in the future.

内生真菌接种方式对青贮玉米幼苗生长的影响

【目的】植物内生真菌是一类重要的微生物资源,对植物生长具有积极的影响。接种方式是内生真菌发挥促生效果的关键环节。探究不同接种方式对内生真菌促生能力的影响,为内生真菌应用提供参考。【方法】以课题组前期从荒漠植物根内生真菌中筛选能促进青贮玉米生长的不同种属的4株内生真菌和印度梨形孢(PI)为供试菌株,采用菌丝片段悬液灌根和菌丝团包根两种方式分别将5株内生真菌接种至青贮玉米盆栽幼苗,30 d后测定接种青贮玉米幼苗的生物学性状,比较不同接种方式对青贮玉米生长的影响。【结果】接种方式对5株内生真菌在青贮玉米幼苗根内菌丝侵染率和幼苗基茎粗均无显著影响,但其他17个与青贮玉米生长相关的性状受接种方式影响显著,主成分分析结果显示5株内生真菌采用菌丝片段悬液灌根方式促生效果均显著优于菌丝团包根,以印度梨形孢和菌株Tm36的两种接种方式得分差值最大。接种印度梨形孢和菌株Tm36的青贮玉米幼苗株高、地上鲜重、地上干重、平均叶面积、叶相对含水量、根鲜重、根表面积、根体积、全株鲜重和全株干重等10个性状在两种接种方式间均有显著差异,其中差异最大的株高、地上鲜重、平均叶面积、根鲜重和全株鲜重等5个指标,菌丝片段悬液灌根接种印度梨形孢增长率是菌丝团包根接种的7.78倍、3.74倍、15.97倍、7.93倍和5.36倍,接种菌株Tm36是8.65倍、4.33倍、11.18倍、16.58倍和7.53倍。【结论】接种方式显著影响内生真菌促生效果,菌丝片段悬液灌根接种方式对青贮玉米幼苗促进生长效果优于菌丝团包根接种方式,推荐使用菌丝片段悬液灌根法。

复方阿莫西林乳房注入剂无菌检查方法的优化

将阿莫西林乳房注入剂无菌检查方法由直接接种法优化为薄膜过滤法。采用肉豆蔻酸异丙酯将样品破乳,与pH 6.0磷酸盐缓冲液混合进行萃取,取萃取后的水相薄膜过滤,采用pH 7.0无菌氯化钠-蛋白胨缓冲液进行冲洗,冲洗完毕加入硫乙醇酸盐液体培养基和胰酪大豆胨液体培养基,定量加入青霉素酶破坏残余的阿莫西林,按照规定条件培养。该方法与直接接种法相比,操作简单,可靠性增强,结果判断准确性提高。

八角无性系对炭疽病抗性的快速测定

[目的]建立一种操作简便、准确可靠的八角无性系炭疽病抗性鉴定方法,为其选育及应用提供理论基础。[方法]采用离体叶片接种法和苗期叶片接种法测定23个八角无性系对炭疽病的抗性水平。[结果]23个八角无性系中,有22个无性系采用2种接种法测定的结果一致,一致性高达95.65%;其中,福进14号和黑叶1号为抗病无性系,福进4号、福进5号和福进6号等9个无性系为中抗无性系,福进2号、福进3号和福进7号等6个无性系为中感无性系,福进8号和福进16号为感病无性系,福进1号、福进11号和福进12号为高感无性系。[结论]2种接种方法均可反应八角无性系抗病性的差异。离体叶片接种法具有操作方便、发病速度快、不伤害植株和耗时短等优点,缺点是鉴定结果可能与实际抗病表现不一致,可以作为八角品系对炭疽病抗性鉴定的重要辅助手段;苗期接种法鉴定结果较为可靠,但缺点是接种后植株落叶严重且难以恢复,需要合适的温、湿度等环境条件。

2-甲基-2-丁醇制备的引发新方法

2-甲基-2-丁醇是一种有用的有机合成中间体。在实验教学中,2-甲基-2-丁醇通常由溴乙烷、镁、丙酮等为原料,无水乙醚为溶剂,在无水、无氧条件下,通过格氏反应制备。溴乙烷与镁在无水乙醚中优先反应制备乙基溴化镁是该反应的关键影响因素。玻璃仪器的干燥、药品的无水处理、镁条的打磨都会影响格氏反应是否能够成功。作者依据多年的实验教学经验,发现了一种格氏反应引发的新方式-接种法。该方法成功率近乎100%。

根瘤菌接种方式对复播大豆生长发育的影响

【目的】筛选出适宜新疆南疆复播大豆生长及产量最佳的接种方式及根瘤菌。【方法】采用3种接种方式(拌种、种肥、滴施)和3种根瘤菌剂(SMH12、T6、SN7-2)对大豆品种绥农35进行田间接种试验,采用裂区试验设计,测定大豆农艺性状、光合特性、产量及产量构成因素的变化。【结果】不同接种方式接种根瘤菌均能不同程度促进南疆复播大豆生长及产量。拌种T6处理对大豆平均主茎节数生长最佳;滴施T6处理对大豆平均株高、LAI生长最佳;滴施SN7-2处理对促进大豆茎粗、LAD、单株粒数、单株荚数生长最佳;拌种SN7-2处理对大豆植株Tr、Gs、Pn、单株粒重及产量为最佳。【结论】产量效果最好的组合为拌种-SN7-2,高达5946.0 kg/hm^(2)。产量较好的组合为滴施-T6达到5888.6 kg/hm^(2),种肥-SN7-2为5461.6 kg/hm^(2),种肥-SMH12为5446.6 kg/hm^(2)。

植物促生菌的功能及在可持续农业中的应用

全球气候变化以及人口增长加剧了农业生产中各种生物(如病原菌)和非生物(如干旱、盐渍、高温等)胁迫,并通过影响植物形态、生理生化特征和代谢功能等阻碍植物的生长、发育和生产力提升,最终影响农作物的产量和品质并严重威胁着农业的可持续发展。随着现代农业的大力发展,有益微生物因其能够改良土壤质量、提高土壤肥力、提升农作物抗胁迫性能和增产提质的功效显著而备受关注。简要概述了植物促生菌(PlantGrowth-PromotingBacteria,PGPB)的种类和施用效应,重点剖析了PGPB产生植物生长激素、固氮作用、加强对营养物质的吸收利用(溶磷、解钾和合成铁载体)、缓解生物和非生物胁迫以及调节植物根系构型和根际微生物群落结构等促生和抗胁迫机制,系统梳理了近年来运用于现代农业中的PGPB菌剂制备和施用方式的前沿科学技术,并进一步讨论了PGPB在未来农业生产中的应用前景以及研究方向。

微生物菌剂处理不同浓度沼液效果

随着沼气工程的发展,沼液处理利用已成为沼气工程可持续发展的瓶颈问题。该文探究了不同微生物菌剂处理方法对不同化学需氧量(chemical oxygen demand,COD)浓度沼液的处理效果。实验结果表明,经过微生物菌剂处理后的沼液感官和理化性质发生明显的变化。低浓度沼液(COD质量浓度为500~2000 mg/L)采用沼液与微生物菌剂一次性投加的方式处理后,沼液有机物和颗粒物大大减少,pH由碱性变为酸性,最大COD去除率为67.6%。高浓度沼液(COD质量浓度为5000~30000 mg/L)采用微生物菌剂一次投加和沼液连续投加的方式处理后,沼液pH变为弱酸性,有机物和悬浮物含量有所下降,但整体变化没有低浓度沼液处理组明显。针对不同浓度的沼液,可采用不同的微生物菌剂处理方法,实现相当的净化效果,可为沼液处理利用提供参考。

苜蓿霜霉病接种方法及染病植株体内酶活性

苜蓿霜霉病(Peronospora aestivalis)是苜蓿上的重要病害之一,严重限制了苜蓿产业的发展。为了探明苜蓿霜霉菌侵染对苜蓿体内保护酶活性的影响及优化苜蓿霜霉菌的接种方法,本研究以紫花苜蓿‘中草3号’(Medicago sativa‘Zhongcao No.3’)为研究对象,进行苜蓿霜霉菌的接种试验,并研究不同染病程度下过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的酶活性,评价接种苜蓿霜霉菌对苜蓿体内保护酶活性的影响。结果表明:4种接种方法中病枝插入法不能获得成功接种的病株,病叶摩擦法、培养箱内喷雾法和培养箱外喷雾法能够得到接种的病株,发病率分别为11.1%、88.9%和25.6%,由此可以看出培养箱内接种法的接种效果最佳。接菌对苜蓿体内酶活性影响显著,苜蓿霜霉菌轻度侵染会导致CAT活性显著增强(P<0.05),较健康苜蓿,苜蓿霜霉菌侵染能够显著增强POD和SOD的活性(P<0.05),但是重度染病情况下与健康苜蓿相比SOD活性差异不显著(P>0.05);PAL活性表现为随着病原菌的侵染程度加深显著下降(P<0.05)。综上表明病原菌侵染会导致苜蓿体内酶活性的变化,本研究可为优化苜蓿霜霉菌的接种方法及通过苜蓿体内酶活性变化筛选抗性品种提供理论参考。

辣椒苗期炭疽菌接种方法

炭疽病是辣椒生产上所面临最严重的病害之一,迫切需要可靠的方法在辣椒发育早期筛选抗病资源。炭疽病菌株CD的分生孢子采用两种接种方法,对辣椒‘ST-8’不同苗龄材料进行接种,通过对发病率、叶片卷曲率以及病斑大小的比较分析最佳的接种方法。结果表明,使用浓度为1×10^(6)个/mL的孢子悬浮液进行针刺点接法效果最佳,有望应用于大规模高通量辣椒抗炭疽病种质筛选。

蚕蛹虫草栽培方法优化试验

不同菌种、培养基配方、接种方法对蛹虫草人工栽培的影响不同。对蚕蛹虫草菌种、培养基配方、接种方法三因素进行优化,以接种后蚕蛹感菌率、出草整齐程度、转色情况等为评价指标进行试验。通过菌株活性检测,筛选出性状稳定的蚕蛹虫草菌种;通过液体菌种培养基配方筛选试验,筛选出发菌快的液体菌种培养基配方;分别采用喷洒、浸泡、注射的方式接种蚕蛹虫草液体菌种,筛选出最优接种方法为注射。

水中五日生化需氧量测定的影响因素

对稀释与接种法测定水中五日生化需氧量的条件进行了分析。水样采集保存、溶解氧浓度、水温、实验样品中的pH值范围、实验过程培养时间、放置时间及培养温度、接种与稀释水和稀释倍数等控制因素的常见问题及对实验结果的影响因素进行了技术性探讨,并结合实际样品在测定分析操作测定过程中出现的问题进行了总结,保证了五日生化需氧量的检测结果的准确性。