一种植物线虫的快速分离方法

目前 ,我国口岸检验检疫机关对于检疫性植物线虫的分离 ,大多数仍然采用贝尔曼漏斗法。该方法的优点是灵敏度高 ,分离物杂质少 ;缺点是分离时间较长 ,大约需要1 0～ 2 4ｈ ,分离效果才达到最佳 ,而且仅限分离活动性的蠕虫形线虫 ,对死虫、不活动的线虫 (如胞囊线虫和根结线虫的雌虫 )则是无效的。我们经过长期的研究与探索 ,找出了一种植物线虫的快速分离方法。将植物线虫的分离时间缩短至 40ｍｉｎ ,并且分离线虫的数量与贝尔曼漏斗法 1 0～ 2 4ｈ的分离数量接近。这种方法适用于目前检疫对象名单上所有的检疫性的线虫分离 ,满足了口岸植物…     

一种快速简便的植物病原真菌基因组DNA提取方法

 尿素提取法是最新报道的一种植物拟南芥基因组DNA的提取方法,但是还没有应用于植物病原真菌DNA的提取。本研究采用改进的4种不同的方法(尿素提取法、CTAB提取法、氯化苄提取法以及SDS-CTAB提取法),分别对5种分类地位不同的植物病原真菌(草莓疫霉病菌、西瓜蔓枯病菌、草莓炭疽病菌、西瓜枯萎病菌和水稻纹枯病菌)的基因组DNA进行提取和比较。结果表明,尿素提取法和SDS-CTAB法均能提取到所有5种真菌的基因组DNA,而尿素提取法提取DNA的效率更高、质量更好,操作步骤更为快速简单。     

A simple generic infection model for foliar fungal plant pathogens

ABSTRACT In this study, a simple generic infection model was developed for predicting infection periods by fungal foliar pathogens. The model is designed primarily for use in forecasting pathogens that do not have extensive epidemiological data. Most existing infection models require a background epidemiological data set, usually including laboratory estimates of infection at multiple temperature and wetness combinations. The model developed in this study can use inputs based on subjective estimates of the cardinal temperatures and the wetness duration requirement. These inputs are available for many pathogens or may be estimated from related pathogens. The model uses a temperature response function which is scaled to the minimum and optimum values of the surface wetness duration requirement. The minimum wetness duration requirement (W(min)) is the number of hours required to produce 20% disease incidence or 5% disease severity on inoculated plant parts at a given temperature. The model was validated with published data from 53 controlled laboratory studies, each with at least four combinations of temperature and wetness. Validation yielded an average correlation coefficient of 0.83 and a root mean square error of 4.9 h, but there was uncertainty about the value of the input parameters for some pathogens. The value of W(min) varied from 1 to 48 h and was relatively uniform for species in the genera Cercospora, Alternaria, and Puccinia but less so for species of Phytophthora, Venturia, and Colletotrichum. Operationally, infection models may use hourly or daily weather inputs. In the case of the former, information also is required to estimate the critical dry-period interruption value, defined as the duration of a dry period at relative humidities <95% that will result in a 50% reduction in disease compared with a continuous wetness period. Pathogens were classified into three groups based on their critical dry-period interruption value. The infection model is being used to create risk maps of exotic pests for the U.S. Department of Agriculture's Animal Plant Health and Inspection Service.     

A simple method for a mini-preparation of fungal DNA

A simple method was established to prepare DNA from fungal mycelia cultured on an agar plate. The fungi tested successfully with this method contained Zygomycetes, Ascomycetes, Basidiomycetes, and Oomycetes. This method did not require any time-consuming steps to crush or digest mycelia or fractionation in a phenol–chloroform mixture. The DNA was easily extracted by immersing and dispersing the mycelial plugs in a specific buffer (200 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid, 200 mM NaCl, 1% n-lauroylsarcosine, pH 8.0), then concentrated by ethanol precipitation. The total time to complete the whole procedure was less than 1 h. The quality and quantity were sufficient for polymerase chain reaction amplification and Southern blot analysis.     

A rapid and simple method for determining fungicide resistance in Botrytis

A simple test based on the germination of conidia of Botrytis on agar media augmented with various fungicides has been developed. Average concentrations causing a 50% reduction of germ-tube growth (EC₅₀) of highly sensitive isolates were determined on 1% malt extract agar (thiophanate-methyl 0.090 ppm; iprodione 0.566 ppm; fludioxonil 0.026 ppm; fenhexamid 0.144 ppm), 1% malt extract agar with 100 ppm salicyl hydroxamic acid (QoI fungicides, viz. trifloxystrobin 0.009 ppm; pyraclostrobin 0.013 ppm; azoxystrobin 0.087 ppm), 0.5% yeast extract agar (boscalid 0.069 ppm) and 0.5% sucrose agar (cyprodinil 0.053 ppm). In order to detect different levels of resistance against these various fungicides, two discriminatory concentrations were identified for each compound. A routine assay method was developed in which drops of a conidial suspension harvested directly from diseased plant material or sporulating cultures were incubated on each of 20 different agar media. Because of a very short time-span of 24–48 h between sample collection and evaluation of results, field-specific information on the occurrence, frequency and types of resistance of Botrytis against common botryticides in soft-fruit production may be generated prior to the main fungicide spray season at blossom time.

     

A simple and effective inoculation method for Phytophthora and fungal species on woody plants

In inoculations carried out on buds, young shoots, and stems of woody plants in Norway, map pins contaminated with the pathogens to be tested proved to be convenient tools. The pins were inserted into the plant tissue and were left standing in the inoculation site throughout the experimental period. In the present investigation, the method was used successfully for one Phytophthora and four fungal species on different host plants. Inoculated plant parts developed clear symptoms, while no symptoms occurred where non‐contaminated pins were used. The advantages of this method are multiple: minimal physical damage to the tissue, inoculation points easily traced due to the relatively large top grip on the needles, and time saving as there is no need to cover with polyethylene or Parafilm after inoculation.     

A rapid method for detecting and quantifying bacterial DNA in rust fungal DNA samples

Bacterial DNA contamination of rust fungal DNA can be a significant problem for sequencing the rust fungus. Sequence assembly is much more difficult if the sequence contigs are mixed with bacterial sequence. A quantitative real-time polymerase chain reaction (qPCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples and the results were compared with those obtained from traditional CFU counts. Real-time PCR showed higher values of DNA contamination than CFU. However, the ranking of samples from low to high for bacterial contamination was consistent between the methods. Reasons for the differences between the methods are discussed. The qPCR assay was tested by adding known quantities of Escherichia coli DNA to Puccinia graminis DNA samples. The assay reliably quantified bacterial contamination at > or = 1.0% of the total sample DNA. When bacterial contamination was <1.0%, fungal DNA also occasionally was amplified, nullifying the quantification measurement. However, primer specificity was not simply the product of the ratio of bacterial DNA to fungal DNA. Bacterial contamination could be quantified below 1.0% if the bacterial DNA concentration was approximately 70 pg/mul or greater. Therefore, spiking the fungal samples with a known concentration of E. coli bacterial DNA successfully eliminated the amplification of fungal DNA, making quantification of contaminating bacterial DNA possible for samples with low contamination levels.     

A rapid method of counting spores of fungal pathogens by infra-red reflectance analysis

A method of counting freshly harvested spores of powdery mildew ( Erysiphe graminis f.sp. hordei ), yellow rust ( Puccinia striiformis ) and brown rust ( P. hordei ) of barley as well as brown rust ( P. recondita ) of wheat, using infra-red reflectance spectrophotometry was investigated. A Neotec 6350 Research Composition Analyser was used to scan spore samples on glass-fibre filter disks in the near infra-red region of the spectrum (1100–2500 nm) and the amount of energy reflected at 1400 different wavelengths recorded. Three wavelengths (1900, 2252 and 2308 nm) that together gave the best multiple correlation with spore populations counted on a haemocytometer slide were selected. Partial regression coefficients for each fungal species were derived by relating reflectance energy values to direct spore counts. Utilizing these and the energy reflected at the three selected wavelengths, it was possible to count spore samples with high precision. Correlations >0.9 between numbers estimated by the instrument and those obtained using a haemocytometer slide (within the range 0–30000 spores) were achieved with all the fungi examined. Application of the technique to smaller, fixed-filter instruments as a routine method of counting spores is discussed.     

一种简单快速的赤霉病菌单孢分离方法 ——平板稀释画线分离法

本文对琼胶平板稀释纯化法进行了改良,建立了一种新方法—平板稀释画线分离法,既保留了其简单快速,操作方便,易于掌握的优点,又解决了可靠性差的缺点,适用于一些不易直接挑取的体积较小,颜色较淡的真菌孢子,如镰刀菌等,和传统分离方法相比节约了大量时间,适合大量样品的分离纯化。     

一种改进的水稻总DNA的快速提取方法

植物总DNA的提取质量是植物分子生物学操作成败的关键,一个优秀的总DNA提取方法往往可以达到事半功倍的效果.本文以水稻叶片为材料,提出了一种改进的植物叶片总DNA快速提取方法,该方法能在35min之内提取到水稻总DNA.电泳结果表明此改进方法提取的总DNA产量与传统CTAB法相当,PCR扩增结果表明其质量可靠,完全可以满足分子生物学操作的要求.     

一种简便易行的栗疫病菌单孢分离方法

介绍了一种分离栗疫病菌单分生孢子的简便易行的方法。此方法可以排除菌丝段的干扰,也适用于分生孢子器中产生微小分生孢子的其他真菌。     

A simple and rapid method for processing tissue infected with plum pox potyvirus for use with specific 3’non-coding region RT-PCR assays1

A rapid, simple method for preparing plant tissue infected with plum pox potyvirus (PPV) using a commercial product known as Gene Releaser is described. The Gene Releaser polymeric matrix method produces plant extracts suitable for PCR amplification without the use of organic solvents, ethanol precipitation, or additional nucleic acid purification techniques. We also describe the development of a PPV-specific amplification assay based on the unique 220 nucleotides present in the 3’non-coding region of the PPV genome. This paper demonstrates the simplicity of the Gene Releaser method combined with the accuracy of the PCR assay for the detection of multiple PPV strains from Spain, France, Greece, Italy, Germany, Egypt, Hungary, and Romania. Amplification of the 3’non-coding region of potato Y potyvirus (PVY) using primers for the 3’non-coding region of the PVY genome was also possible with Gene Releaser preparation of viruliferous Myzus persicae to demonstrate the potential usefulness of this work for PPV detection from aphids.     

Cold plasma: a potential new method to manage postharvest diseases caused by fungal plant pathogens

Development of alternative, chemical‐free approaches for control of postharvest fungi on a commercial scale has become a challenge for plant pathologists in recent years. Although there are several established techniques such as heat that are used as postharvest treatments, they often have disadvantages, including alteration of food quality due to physiological responses to the treatment, or environmental pollution. A promising new postharvest treatment is cold plasma, which is a gas‐derived mix of atoms, excited molecules and charged particles. Cold plasma has no known adverse effects on fresh produce or the environment. It is an established technology in the medical field and has been demonstrated to successfully control bacterial pathogens that cause food safety issues. This review focuses on the potential of cold plasma technology for postharvest disease control, especially those caused by fungi. An overview of plasma generation systems is provided, and in vivo and in vitro research is reviewed to consider benefits, limitations and research gaps in the context of cold plasma as a potential method for controlling postharvest fungal pathogens. Finally, recommendations are provided for the application of this technology in commercial facilities.     

水稻干尖线虫快速分子检测技术研究

根据水稻干尖线虫的rDNA-ITS序列,设计出水稻干尖线虫的特异性引物,利用PCR技术对水稻干尖线虫(Aphelenchoides besseyi)部分rDNA-ITS1和部分5.8S基因核苷酸序列进行特异性扩增。实现了单条活的或4%的甲醛(FG)固定的水稻干尖线虫的快速检测。     

Toxicity of fungal endophyte secondary metabolites to plant parasitic nematodes and soil-borne plant pathogenic fungi

Fungi isolated from the cortical tissue of surface sterilized tomato roots collected from field plots produced secondary metabolites in nutrition broth that were highly toxic toMeloidogyne incognita. Especially strains ofFusarium oxysporum were highly active with 13 of 15 strains producing culture filtrates toxic to nematodes. The mechanism of action of the toxic metabolites produced by the non-pathogenicF. oxysporum strain 162 with proven biological control ofM. incognita in pot experiments was investigated. These metabolites reducedM. incognita mobility within 10 min of exposure. After 60 min, 98% of juveniles were inactivated. Juveniles were initially inactivated within a few minutes of exposure, but with exposure of 5 h 50% of the juveniles were dead and 24 h exposure resulted in 100% mortality. In a bioassay with lettuce seedlings metabolite concentrations > 100 mg/l reduced the number ofM. incognita juveniles on the roots comparing to the water control. TheF. oxysporum toxins were highly effective towards sedentary parasites and less effective towards migratory endoparasites. Nonparasitic nematodes were not influenced at all. Metabolites of strain 162 also reduced significantly the growth ofPhytophthora cactorum, Pythium ultimum andRhizoctonia solani in vitro. Secondary metabolites of endophytic fungi on plant-parasitic nematodes and soil-borne fungi should be considered for control of plant parasitic nematodes and plant pathogenic fungi. The results also show the need for proper selection of target nematodes inin vitro bioassays.     

Properties of viruses of the potyvirus group. 1. A simple method to purify bean yellow mosaic virus,pea mosaic virus,lettuce mosaic virus and potato virus YN

Bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus, and potato virus Y^N were purified by homogenizing and clarifying infected leaves in a mixture of 0.1 M tris-thioglycollic acid buffer pH 9, carbon tetrachloride and chloroform, followed by differential centrifuging applying moderate centrifugal forces.Samenvatting Het bonescherpmozaïekvirus B25, het erwtemozaïekvirus E198, het slamozaïekvirus en het aardappelvirus Y^N konden worden gezuiverd door geïnfecteerde bladeren te vermalen in een mengsel van 0,1 M tris-thioglycolzuur pH 9, tetrachloorkoolstof en chloroform, en de geklaarde suspensie vervolgens differentiëel te centrifugeren bij matige centrifugaalkrachten.     

A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.     

Polygalacturonase inhibiting proteins fromAllium porrumL. and their role in plant tissue against fungal endo-polygalacturonases

Five endo-polygalacturonases (PGs), three produced in culture filtrate byFusarium moniliforme, Sclerotium cepivorumandBotrytis aclada,respectively, and two (one acidic and one basic isoform) obtained fromSclerotinia sclerotiorumsoybean infected hypocotyls, were purified in order to characterize the activity of polygalacturonase inhibitor(s) (PGIP(s)) from leek stalk tissue (Allium porrumL.). Three apparently different PGIPs (PGIP-I, PGIP-II and PGIP-III) were purified from the leek tissue. The two more abundant PGIPs (PGIP-I and PGIP-III), although possessing similar pIs of about 6.5, differed in chromatographic behaviour, their molecular mass (39 and 42 kDa, respectively), and specific activity when assayed with the fungal endo-PGs. In addition, PGIP-I was solubilized from tissue homogenate with a low-salt buffer whilst PGIP-III needed a high-salt buffer for extraction (behaving as an ionically wall-bound protein). PGIP-II had very similar properties to PGIP-I, but was extracted using the high-salt buffer. The purified PGIPs and the crude leek extract showed similar inhibition activity patterns against the five fungal endo-PGs. The maximum inhibition activity was observed against the basic endo-PG fromS. sclerotiorum,followed by the acidic endo-PG ofS. sclerotiorumand the endo-PG fromB. aclada.In contrast, no inhibition of endo-PGs fromS. cepivorumandF. moniliformewas observed. Four different concentrations of the five fungal endo-PGs were incubated separately with slices of leek stalk, and the galacturonides released in the incubation mixture were measured. At every level used the endo-PGs ofF. moniliformeandS. cepivorumshowed the maximum activity in uronide releasing. The endo-PGs ofS. sclerotiorum(acidic PG) andB. acladawere active only when high levels were used while the basic endo-PG ofS. sclerotiorumwas not active in combustion with any level of PGIP. These results indicate that a close relationship exists between PGIP activityin vitroand the ability of PGIP to protect leek tissue from endo-PG degradation.