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.     

Inhibition of fungal spore adhesion by zosteric Acid as the basis for a novel, nontoxic crop protection technology

ABSTRACT To explore the potential for nontoxic crop protection technologies based on the inhibition of fungal spore adhesion, we have tested the effect of synthetic zosteric acid (p-(sulfo-oxy) cinnamic acid), a naturally occurring phenolic acid in eelgrass (Zostera marina L.) plants, on spore adhesion and infection in two pathosystems: rice blast caused by Magnaporthe grisea and bean anthracnose caused by Colletotrichum lindemuthianum. We have shown that zosteric acid inhibits spore adhesion to model and host leaf surfaces and that any attached spores fail to develop appressoria, and consequently do not infect leaf cells. Low concentrations of zosteric acid that are effective in inhibiting adhesion are not toxic to either fungus or to the host. The inhibition of spore adhesion in the rice blast pathogen is fully reversible. On plants, zosteric acid reduced (rice) or delayed (bean) lesion development. These results suggest that there is potential for novel and environmentally benign crop protection technologies based on manipulating adhesion.     

Dynamics of cercospora leaf spot disease determined by aerial spore dispersal in artificially inoculated sugar beet fields

Cercospora beticola is one of the most important fungal pathogens of sugar beet, causing cercospora leaf spot (CLS) disease. Due to the decreasing efficacy of various fungicides caused by resistance traits, the development of a sustainable disease management strategy has become more important. Therefore, detailed knowledge about the epidemiology of the pathogen is crucial. Until now, little was known about the spatiotemporal dispersal of C. beticola spores from the primary inoculum source. Rapid detection of C. beticola spores could facilitate a more precise and targeted disease control. Therefore, a TaqMan real-time PCR assay for detection and quantification of C. beticola spores caught with Rotorod spore traps was established. In 2016 and 2017, field trials were conducted to monitor C. beticola aerial spore dispersal and disease development within an inoculated field and in the adjacent noninoculated area. With the established detection method, C. beticola spores were successfully quantified and used as a measure for aerial spore dispersal intensity. The analysis of the spatiotemporal spread of C. beticola spores revealed a delay and decrease of aerial spore dispersal with increasing distance from the inoculated area. Consequently, disease incidence and severity were reduced in a similar manner. These results imply that spore dispersal occurs mainly on a small scale within a field, although long distances can be overcome by C. beticola spores. Moreover, secondary aerial spore dispersal from sporulating leaf spots seems to be the main driver for CLS disease development. These results provide an important basis for further improvement of CLS control strategies.     

Smoke puffs as models for the study of spore dispersal in and above a cereal crop

Samenvatting De verspreiding van roestsporen door turbulente luchtbewegingen wordt geïllustreerd met behulp van rookwolkjes. Rookwolkjes werden geproduceerd nabij het actieve buitenoppervlak van een gerstgewas. De bewegingen van de rookwolkjes werden gefilmd. Iedere kwart seconde werd de op de film zichtbare buitenomtrek van een rookwolkje op een tekening vastgelegd (Fig. 1 t/m 10). De verplaatsing van het zwaartepunt van ieder rookwolkje kon een aantal seconden gevolgd worden (Fig. 11). De waarnemingen ondersteunen de gangbare opvattingen over de verspreiding van uredosporen binnen de gewasruimte en vanuit het gewas naar de, turbulente lucht daarboven.     

Effect of environmental conditions on spore production by Fusarium verticillioides, the causal agent of maize ear rot

Silk infection by Fusarium verticillioides is caused by conidia produced on maize crop residues and results in kernel infection and consequent accumulation of fumonisins. Studies were carried out in both controlled and field conditions to understand the dynamics of sporulation on maize residues. The effect of temperature (5°C to 45°C) and incubation time (3 to 41 days) on spore production on maize meal agar was described by a logistic model that accounted for 85% of variability. The rate parameter depended on the length of incubation and the asymptote on temperature. Maximum sporulation occurred at 27°C, with a progressive increase between 5°C and 27°C and then a rapid decline, with no sporulation at 45°C. Fusarium verticillioides strains from different geographic origins showed different sporulation capabilities, with similar optimum temperatures. Pieces of stalk residues inoculated with F. verticillioides and placed above the soil between rows of maize crops, in 2003 to 2005, produced conidia continuously and abundantly for some weeks, particularly during the period after silk emergence, with an average of 1.59 × 10⁷ conidia g⁻¹ of stalk, over a wide range of environmental conditions. Sixty-seven percent of variability of the spore numbers found on stalks was accounted for by a multiple regression model. Precipitation (rain or overhead irrigation) in the 14 days before stalk sampling decreased the number of spores, whilst the number of days with conducive conditions of moisture (i.e. days with rainfall, average relative humidity >85% or vapour pressure deficit <4 hPa) and greater degree-days (base 0°C) in the 14 and 3 days before sampling, respectively, increased sporulation.     

基于病菌孢子捕捉和real-time PCR技术的田间空气中小麦白粉病菌孢子动态监测及病情估计模型研究

利用Burkard定容式孢子捕捉器结合real-time PCR定量技术,分别对种植高抗、中感和高感白粉病小麦品种的田间空气中白粉病菌分生孢子浓度进行监测,结果表明,real-time PCR定量与传统的显微观察计数两种方法测得的孢子浓度呈显著正相关(P≤0.01),且两种病菌孢子计数方法在同一抗性品种上监测到的孢子浓度动态相近。此外,两种方法测得的孢子浓度与各气象因子的相关性分析结果一致,空气中的白粉病菌孢子浓度主要与空气相对湿度显著正相关。在此基础上,利用两种方法测定的田间空气中白粉病菌孢子浓度分别建立了基于累积孢子浓度的田间病情估计模型。分析发现,基于两种孢子浓度测定方法建立的病情估计模型间无显著性差异,表明real-time PCR定量技术测定的孢子浓度在构建白粉病病情估计模型上具有一定可行性。该结果为real-time PCR定量技术与病菌孢子捕捉技术相结合用于小麦白粉病的监测和预测提供理论依据。     

Relationship between the fungal complex causing Fusarium head blight of wheat and environmental conditions

ABSTRACT Over 4 years, the environmental conditions and the causal agents of Fusarium head blight (FHB) disease of wheat were determined in field sites in four European countries: Hungary, Ireland, Italy, and the United Kingdom. Polymerase chain reaction-based methods were used to detect each species causing FHB and quantify its DNA (as a measurement of fungal abundance) in the samples. Canonical correspondence analysis (CCA) was used to determine the relationship of the incidence and abundance of each species with weather variables. CCA indicated that little variability in the species prevalence data was explained by the weather variables. In contrast, a greater proportion of variability in abundance data was accounted for by the weather variables. Most samples contained two or more species and statistical analysis suggested that these species tended to coexist at field sites. CCA also indicated that there were differences in the relationships of the prevalence and abundance of the six FHB species with environmental variables. Fusarium poae was associated with relatively drier and warmer conditions, whereas F. graminearum was associated with warmer/humid conditions. F. avenaceum and F. culmorum were both associated with niches of cooler/wet/humid conditions. Two Microdochium species were associated with regions of relatively cool/moderate temperatures and frequent rainfalls of short duration. The results also suggested that environmental conditions differentially affect the infection and colonization processes, and the comparative abundance of the six species.     

Hyperspectral signal decomposition and symptom detection of wheat rust disease at the leaf scale using pure fungal spore spectra as reference

This study establishes a method to detect and distinguish between brown rust and yellow rust on wheat leaves based on hyperspectral imaging at the leaf scale under controlled laboratory conditions. A major problem at this scale is the generation of representative and correctly labelled training data, as only mixed spectra comprising plant and fungal material are observed. For this purpose, the pure spectra of rust spores of Puccinia triticina and P. striiformis, causal agents of brown and yellow rust, respectively, were used to serve as a spectral fingerprint for the detection of a specific leaf rust disease. A least-squares factorization was used on hyperspectral images to unveil the presence of the spectral signal of rust spores in mixed spectra on wheat leaves. A quantification of yellow and brown rust, chlorosis and healthy tissue was verified in time series experiments on inoculated plants. The detection of fungal crop diseases by hyperspectral imaging was enabled without pixel-wise labelling at the leaf scale by using reference spectra from spore-scale observations. For the first time, this study shows an interpretable decomposition of the spectral reflectance mixture during pathogenesis. This novel approach will support a more sophisticated and precise detection of foliar diseases of wheat by hyperspectral imaging.     

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.     

孢子捕捉仪在植物病害监测中的应用

作为一种孢子取样和监测工具,孢子捕捉仪的应用实现了空气中病原菌孢子的捕捉、计数和识别等功能,使许多流行性病害得以提前预警,及时精准施策,从而为作物高产稳产起到了关键性作用。本文在对各种类型孢子捕捉仪自身特点和实际应用效果对比分析的基础上,以孢子捕捉技术的发展为主线,详细介绍了常见孢子捕捉仪的原理、特点、应用范围和捕捉后分析方法,综述了孢子捕捉仪在植物病害流行研究和生产实践中的应用情况,以及如何量化与鉴定所捕捉病原菌孢子的方法。为更好地实现田间病害监测及防治策略制订提出了孢子捕捉仪今后改进的方向,旨在使该类仪器在病害监测方面更好地服务于农林生产。     

On the ontogeny of the fungus spore

Fungus spores can be borne endogenously in sporangia, gametangia or asci, or exogenously on basidia, conidiophores or sporogenic cells. Exogenous spores can be produced by sprouting (blastospores), by fission (arthrospores) or by an intermediate manner (sprouting followed by fission). This has to be considered especially in the classification of the imperfect fungi.Samenvatting Schimmelsporen kunnen zowel endogeen als exogeen ontstaan. Cellen met een endogene sporevorming heten sporangia. Wanneer aan de sporevorming een reductiedeling vooraf gaat, worden de sporogene cellen asci genoemd. Exogene sporevorming geschiedt door afgrenzing door middel van een celwand (arthrosporen en aleuriosporen) of door knopvorming of wel spruiting (blastosporen).Hiertussen komen overgangen voor: na een knopvorming kan het knopgedeelte zich van de moedercel afgrenzen door een dwarswand. Er wordt voorgesteld bij de indeling van de imperfecte fungi met dergelijke tussenvormen rekening te houden, zoals dit bij de indeling van de gisten geschiedt. Basidiosporen zijn blastosporen die na een reductiedeling op een gespecialiseerde moedercel ontstaan.     

硼抑制灰霉病菌孢子萌发机制的初步研究

 本文研究了硼对灰霉病菌孢子萌发的影响并对其抑制机理作了进一步的分析。用0.1%硼处理灰霉病菌孢子后,显著地抑制了孢子萌发和芽管伸长。与不处理的对照相比,0.1%硼使灰霉病菌孢子细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性逐渐下降,还原型谷胱甘肽(GSH)和抗坏血酸(ASA)含量逐渐降低,丙二醛(MDA)、过氧化氢(H₂O₂)含量和超氧阴离子(O₂^·)产生速率上升。此外,DCHF-DA荧光染色结果也进一步证明硼处理造成了灰霉病菌孢子细胞内活性氧的积累。本试验结果初步表明,0.1%硼处理使细胞内活性氧清除系统破坏,膜脂过氧化作用加强,是抑制灰霉病菌孢子萌发和芽管伸长的重要原因。     

生态系统服务功能偏好异质性研究——基于渭河流域水资源支付意愿的分析

流域治理政策的建立需要充分考虑公众对环境物品的偏好及偏好的异质性问题。文中运用选择实验法(Choice Experiment),以渭河流域生态系统服务功能改善为例展开实证研究,结合Mixed logit模型和潜类别模型从效用和支付意愿的视角揭示了公众对生态系统服务功能的偏好异质性。研究发现,公众对渭河流域生态系统服务功能具有较强的偏好,且存在显著的偏好异质性;潜类别模型中偏好正常型消费者占据大部分消费者总数,生态导向型消费者在水质等方面的支付意愿明显较高,而价格敏感型消费者对所有生态系统服务功能的支付意愿明显低于其他两类消费者。总体来看,居民对水质、水量等生态系统服务功能具有较强的支付意愿,而对自然景观和生态旅游具有较低的支付意愿。     

新经济地理学框架下环境问题研究综述

基于新经济地理学研究框架,对该领域关于环境问题的研究予以梳理,在回顾其发展历程的基础上,总结出当前新经济地理学框架下,环境问题研究的主要议题涉及环境外部性与集聚效应、对经济活动空间分布的影响,以及环境规制与响应等。此外,从研究方法的角度,整理出在新经济地理学框架下涉及环境因素分析的模型及方法,并举例详述了引入环境因素的多种经典新经济地理模型及其结论。最终结合当前研究进展,总结了该领域的演化分析、环境CGE模型和微观异质性分析等研究前沿,为环境问题在新经济地理学领域的深入研究提供了新的思路。     

小麦白粉病菌分生孢子田间传播的初步研究

2015年度采用定容式孢子捕捉器对田间空气中小麦白粉病菌分生孢子传播的初步研究表明,在病害发生初期病菌分生孢子捕捉量比较低,但随着菌源中心病害的逐渐加重,病菌分生孢子在距菌源中心20 m和40 m远处捕捉量随之增大。线性弧度相关分析结果发现,距菌源中心北向20 m和40 m处的孢子捕捉量与风向存在显著正相关性;距菌源中心不同距离处的孢子捕捉量之间均存在极显著正相关性,且在相同方向上,距菌源中心20 m处孢子捕捉量显著高于40 m处孢子捕捉量。     

A new spore precipitator with polarized dielectric insulators for physical control of tomato powdery mildew

ABSTRACT In an attempt to physically protect greenhouse tomato plants from the powdery mildew fungus Oidium neolycopersici, we developed a new electrostatic spore precipitator in which a copper wire conductor is linked to an electrostatic generator and covered with a transparent acrylic cylinder (insulator). The conductor was negatively charged by the generator, and the electrostatic field created by the conductor was used to dielectrically polarize the insulator cylinder. The dielectrically polarized cylinder also produced an electrostatic force without a spark discharge. This force was directly proportional to the potential applied to the conductor and was used to attract conidia of the pathogen. The efficacy of this spore precipitator in protecting hydroponically cultured tomato plants from powdery mildew was evaluated in the greenhouse. The hydroponic culture troughs were covered with a cubic frame installed with the spore precipitator, and the disease progress on precipitator-guarded and unguarded seedlings was traced after the conidia were disseminated mechanically from inoculum on tomato plants. Seedlings in the guarded troughs remained uninfected during the entire experiment, in spite of rapid spread of the disease to all leaves of the unguarded seedlings.     

真菌源几丁质酶在植物抗真菌病害中的应用

几丁质酶可以降解大多数真菌细胞壁,阻止或中断真菌在植物体内的侵染、定殖和扩展,在抗真菌病害的研究方面受到广泛关注。本文综述了真菌源几丁质酶的特性,对真菌的抑制作用机制,转几丁质酶基因植株的抗性评价,产几丁质酶菌株和相关制剂在生物防治方面的应用,并对其在生产上的发展前景进行了展望。