Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Production and Evaluation of Monoclonal Antibodies for the Detection of Pythium sulcatum in Soil

Monoclonal antibodies (MAbs) were raised against surface antigens from Pythium sulcatum. The immunogens were prepared from salt extractable cell wall protein to produce monoclonal antibodies. The MAbs showed high specificity to seven P. sulcatum isolates among 26 species of soil‐borne fungi. Weak cross‐reactivities were observed with Pythium aristosporum, Pythium myriotylum, and Pythium zingiberum in indirect enzyme‐linked immunosorbent assay (ELISA), but no reaction was obtained in Western blot analysis. The MAbs recognized glycoproteins in cell wall. Pythium sulcatum was detected in naturally infected carrot tissues and soil using indirect competition ELISA.     

Serological detection of red rot disease initiation stages of microbial pathogen, Pythium porphyrae (Oomycota) on Porphyra yezoensis

Pythium porphyrae (Oomycota), a pathogen causing red rot diseasein Porphyra spp., can at present only be detected when colonizationof the host thallus has already occurred and so it is often too late to takeappropriate disease control measures. The paper presents an account of an effective methdology for early detection of the disease. Since Py.porphyrae zoospores are the primary means of pathogen dispersal,polyclonal antibodies (Pabs) were raised against the surface components ofzoospores and encysted zoospores. Using these Pabs the disease initiationstages of the Pythium porphyrae were detected on the surface of Porphyra thalli by immunofluorescence assay. The specificity of theseantibodies and the efficacy of immunofluorescence assay in the detectionof red rot disease are discussed.     

A simple in-vitro 'wet-plate' method for mass production of Phytophthora nicotianae zoospores and factors influencing zoospore production

A simple in-vitro ‘wet-plate’ method for mass-producing Phytophthora nicotianae zoospores at ≥ 1.0 × 10⁶ zoospores/ml is described. Temperature critically affected zoospore production; 22 °C was optimum, while 36 °C was completely inhibitory. Zoospores being the most important propagule of P. nicotianae, temperature of recycled irrigation water may be manipulated to reduce diseases in irrigated nursery crops.     

膜下滴灌条件下滴水量和滴水频率对棉田土壤水分分布及水分利用效率的影响

通过两年的田间试验,研究了滴水量和滴水频率对膜下滴灌棉田土壤水分分布及棉花水分利用效率的影响.结果表明:从整个生育期来看,当滴水量(375 mm)相同时,高频滴灌(每3天1次)处理0～20 cm土层含水率较高而深层土壤湿润不够;低频滴灌(每10天1次)处理有利于水分的下渗和侧渗,深层土壤含水率较高,但水分补给不及时,表层土壤偏低;总体上中频滴灌(每7天1次)处理有利于水分在土壤剖面中的均匀分配.当滴水频率相同时,滴水量越大,土壤含水率越高,40 cm以下土层含水率也越高.不同处理的棉田耗水规律基本一致,苗期较低,平均不高于1.7 mm·d-1,蕾期开始上升至花铃期达到最高,日均耗水量可达8.7 mm·d-1,吐絮期回落到1.0 mm·d-1左右.总耗水量与降水和滴水量密切相关,而与滴水频率无关;滴水频率对棉花水分利用效率无显著影响,但水分利用效率随滴水量的增大而显著降低.少量滴灌(300 mm)虽然可以获得较高的水分利用效率,但减产严重,过量滴灌(450mm)无显著增产效应,水分浪费严重.在当地棉田自然条件下,采用中量(375 mm)+中低频(每7天或10天1次)的滴灌模式为宜.     

膜下滴灌条件下滴水量和滴水频率对棉田土壤水分分布及水分利用效率的影响

通过两年的田间试验,研究了滴水量和滴水频率对膜下滴灌棉田土壤水分分布及棉花水分利用效率的影响.结果表明： 从整个生育期来看,当滴水量(375 mm)相同时,高频滴灌(每3天1次)处理0～20 cm土层含水率较高而深层土壤湿润不够;低频滴灌(每10天1次)处理有利于水分的下渗和侧渗,深层土壤含水率较高,但水分补给不及时,表层土壤偏低;总体上中频滴灌(每7天1次)处理有利于水分在土壤剖面中的均匀分配.当滴水频率相同时,滴水量越大,土壤含水率越高,40 cm以下土层含水率也越高.不同处理的棉田耗水规律基本一致,苗期较低,平均不高于1.7 mm·d^-1,蕾期开始上升至花铃期达到最高,日均耗水量可达8.7 mm·d^-1,吐絮期回落到1.0 mm·d^-1左右.总耗水量与降水和滴水量密切相关,而与滴水频率无关;滴水频率对棉花水分利用效率无显著影响,但水分利用效率随滴水量的增大而显著降低.少量滴灌(300 mm)虽然可以获得较高的水分利用效率,但减产严重,过量滴灌(450 mm)无显著增产效应,水分浪费严重.在当地棉田自然条件下,采用中量(375 mm)+中低频(每7天或10天1次)的滴灌模式为宜.     

亏缺灌溉对棉花生长和水分利用效率的影响研究进展

棉花是世界上最主要的农作物之一.随着全球水资源的日益紧张,灌溉用水将成为限制棉花生产的主要因素.亏缺灌溉是一种低于作物正常腾发量的灌溉方式,可以在保证棉花产量和品质的前提下提高水分利用效率,是一种有效的节水灌溉方式.本文综述了亏缺灌溉对棉花生长和水分利用效率的影响.亏缺灌溉可以通过促进棉花由营养生长向生殖生长转化,降低...     

基于恒水位蒸发皿蒸发量的膜下滴灌棉花灌溉指标

为探索新疆膜下滴灌棉花简易方便的高效灌溉指标,于2008-2009年在乌鲁木齐开展了2个生长季的人工控水试验.在棉花蕾期和花铃期均设2个灌水周期和2个灌水水平,分析了不同水分处理对棉花产量、耗水量和水分利用效率的影响.结果表明: 各处理的棉花耗水过程与蒸发皿蒸发量具有较高的相关性,高产棉田\[2008年处理T₄(蕾期和花铃期灌水周期分别为10和7 d,相应灌水定额分别为30.0和37.5 mm)和2009年处理T₁(蕾期和花铃期灌水周期均为7 d,相应灌水定额分别为22.5和37.5 mm)\]苗期、蕾期、花铃期和吐絮期的蒸发皿-作物系数(K_p)分别为0.29～0.30、0.52～0.53、0.74～0.88和0.19～0.20;2008年处理T₄的产量(5060 kg·hm^-2)和水分利用效率(1.00 kg·m^-3)最高,2009年处理T₁的产量(4467 kg·hm^-2)和水分利用效率(0.99 kg·m^-3)最高;蕾期蒸发皿7和10 d的平均累积蒸发量分别为40～50和60～70 mm,花铃期蒸发皿7 d的累积蒸发量为40～50 mm.在新疆棉区灌45 mm出苗水、苗期和吐絮期不灌水,蕾期和花铃期当蒸发皿蒸发量达到45～65和45 mm时开始灌溉,灌水定额通过阶段累积蒸发量与蒸发皿-作物系数K_p(蕾期、初花期、盛花期和末花期分别取0.5、0.75、0.85和0.75)相乘确定时,在获得高产的同时可节约灌溉水资源,提高水分利用效率,可以作为当地膜下滴灌棉田简易方便的高效灌溉指标.     

滴灌条件下温室番茄需水量估算模型

基于修正后的Pnman-Monteith方程,通过分析作物系数与积温的关系,构建了基于常规气象资料的滴灌条件下温室番茄需水量估算模型,并分别采用2009年5月2-13日(开花坐果期)和6月9-20日(成熟采摘期)2个时段内的实测蒸腾量和实测棵间土壤蒸发量对模型模拟结果进行验证.结果表明:修正后的Penman-Monteith方程适用于温室参考作物需水量(ET0)的计算;温室番茄作物系数与积温呈抛物线关系;所建需水量模型模拟值的平均相对误差小于10%,可用于估算滴灌条件下温室番茄需水量.     

A PCR-RFLP assay for identification and detection of Pythium myriotylum, causal agent of the cocoyam root rot disease

Aim: Development of a PCR‐RFLP assay that could reliably distinguish strains of Pythium myriotylum that are pathogenic to cocoyam from nonpathogens, as well as in planta detection of the pathogen. Methods and Results: Sequences of the internal transcribed spacer regions of nuclear ribosomal DNA (rDNA‐ITS) containing ITS1 and ITS2 of P. myriotylum isolates from cocoyam and other hosts were aligned and a restriction map was generated. rDNA‐ITS alignment report revealed a new single nucleotide polymorphism (SNP; thymine/cytosine) downstream to previously published SNP (guanine/adenine) between isolates of P. myriotylum that are pathogenic to cocoyam and nonpathogenic strains. This new SNP is within the restriction site of the endonuclease AarI. Based on this SNP, a PCR‐RFLP assay was developed for specific detection of P. myriotylum. The PCR amplicons of all isolates of P. myriotylum that infect cocoyam were cleaved by AarI, resulting to two bands (600/400 bp); but those from other hosts showed a single band (1000 bp), confirming the presence and specificity of the AarI restriction site. Also, the assay was effective in in planta detection of the pathogen on infected cocoyam roots without prior isolation of a pure culture. Conclusion: A PCR‐RFLP method was developed that differentiates isolates of P. myriotylum that are pathogenic to cocoyam from nonpathogens as well as from other fungi commonly found in the cocoyam rhizosphere. Significance and Impact of the Study: Early and rapid detection of the pathogen could be of great importance in certifying planting materials as disease‐free, enhancing sustainable management practices and limiting economic losses.     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

调亏灌溉对冬小麦耗水特性和水分利用效率的影响

以高产中筋冬小麦品种济麦22为材料,在山东兖州小孟镇史王村进行田间试验,研究了调亏灌溉对冬小麦耗水特性和水分利用效率的影响.结果表明：在全生育期降水228 mm条件下,W₁（土壤相对含水量：播种期80%+拔节期70%+开花期70%）和W₄（土壤相对含水量：播种期90%+拔节期85%+开花期85%）处理总耗水量高于W₀（土壤相对含水量：播种期80%+拔节期65%+开花期65%）、W₂（土壤相对含水量：播种期80%+拔节期80%+开花期80%）和W₃（土壤相对含水量：播种期90%+拔节期80%+开花期80%）处理,W₁和W₄处理间无显著差异;W₁处理增加了0～200 cm土层土壤贮水消耗量,降低了小麦拔节至开花期的耗水模系数,提高了开花至成熟期的耗水模系数;W₄处理在开花至成熟期、拔节至开花期的耗水量和耗水模系数均较大.调亏灌溉条件下,W₀处理水分利用效率较高,但产量最低;随灌溉量增加,其他处理水分利用效率呈先增加后降低的趋势.耗水量最高的W₁和W₄处理产量也最高,W₁处理灌溉水利用效率和灌溉效益均高于W₄处理,为本试验条件下高产节水的最佳处理.     

Gold immunochromatographic assay for simultaneous detection of carbofuran and triazophos in water samples

Using a simple test for rapid identification and quantification of pesticide multiresidues in food and environmental samples is a long-cherished approach for practical monitoring purposes. Here two gold-based lateral-flow strips (strip A and strip B) were investigated for simultaneous detection of carbofuran and triazophos. For the strip A format, a bispecific monoclonal antibody (BsMcAb) against both carbofuran and triazophos was employed to prepare the immunogold probe. For the strip B format, anti-carbofuran monoclonal antibody (McAb) and anti-triazophos McAb separately labeled with colloidal gold were combined as detector reagents. By comparison of visual results from pesticide standard tests between the two formats, the strip B assay manifested higher sensitivities for both pesticides. Analysis of spiked water samples by the preferable strip indicated that the detection limits for carbofuran and triazophos were 32 and 4 μg/L, respectively. The strength of the portable one-step strip assay was in the simultaneous screening for two pesticides within a short time (8-10 min) without any equipment.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

有限供水下冬小麦全程耗水特征定量研究

为明确冬小麦不同水分条件下全生育过程日耗水及阶段耗水特征,在北京地区利用蒸渗仪系统连续监测了几种灌溉处理(W4:起身水+孕穗水+开花水+灌浆水;W2:拔节水+开花水;W1:拔节水;W0:无灌水)耗水动态变化。结果表明:冬小麦全生育期的耗水动态可分为3个阶段:(1)播种至11月底的冬前阶段,这个阶段日耗水量波动明显,一般低于3 mm/d;(2)12月上旬至来年2月底的冬季阶段,这个阶段日耗水量低于0.4 mm/d,且波动很小;(3)3月初小麦返青至收获的春后生长阶段,这个阶段日耗水量总体上是一个先升高后降低的过程,但波动很大,每次灌水都会引起1个日耗水高峰的出现。耗水日变化呈单峰或双峰曲线,高峰出现在正午前后,高峰值因灌水处理而有明显差异,灌水多则耗水峰值显著升高,而夜间耗水量及其在不同处理间差异均很小。拔节至成熟期是冬小麦耗水的主要时期,该期耗水占总耗水60%以上。减少灌溉会增加土壤贮水消耗,但降低了总耗水量。综合比较表明,在有限灌溉下,拔节水和开花水组合是高产和高水分效率相统一的灌溉模式。     

灌溉对土壤盐分的影响及微咸水利用的模拟研究

在土地利用分析模型PS123的基础上,以土壤盐分主要影响作物水分吸收为突破口,将盐分对作物生长的影响结合到模型中,以使模型更加适合于盐渍化土地和微咸水灌溉的使用。并运用田间试验对模型进行了验证,模拟结果较好。利用模型对微咸水不同的灌溉方案对土壤盐分的影响进行分析,提出了合理的灌溉方案。盐渍化地区地下水位埋深应控制在2．0～2．5m以下,微咸水灌溉宜采用少次多量的措施进行较大定额灌溉,且不能连续灌溉。