Exploiting generic platform technologies for the detection and identification of plant pathogens

The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.     

Recent developments in pathogen detection arrays: implications for fungal plant pathogens and use in practice

ABSTRACT The failure to adequately identify plant pathogens from culture-based morphological techniques has led to the development of culture-independent molecular approaches. Increasingly, diagnostic laboratories are pursuing fast routine methods that provide reliable identification, sensitive detection, and accurate quantification of plant pathogens. In addition, since plants or parts thereof can be infected by multiple pathogens, multiplex assays that can detect and quantify different pathogens simultaneously are highly desirable. Technologies that can meet these requirements, especially those involving polymerase chain reaction, are being developed and implemented in horticultural and agricultural practice. Currently, DNA array technology is the most suitable technique for multiplex detection of plant pathogens. Recently, a quantitative aspect was added to this technology, making DNA arrays highly attractive for various research and practical applications. Here, we review the most important recent advances in molecular plant pathogen diagnostics, with special attention to fungal molecular diagnostics. In addition to their applicability in practice, the different criteria that have to be fulfilled for developing robust detection procedures that can routinely be used by diagnostic laboratories are discussed.     

Chlorophyll fluorescence for quantification of fungal foliar infection and assessment of the effectiveness of an induced systemic resistance activator

Chlorophyll fluorescence imaging was used to follow infections of Nicotiana benthamiana with the hemibiotrophic fungus, Colletotrichum orbiculare. Based on F_v/F_m images, infected leaves were divided into: healthy tissue with values similar to non-inoculated leaves; water-soaked/necrotic tissue with values near zero; and non-necrotic disease-affected tissue with intermediate values, which preceded or surrounded water-soaked/necrotic tissue. Quantification of F_v/F_m images showed that there were no changes until late in the biotrophic phase when spots of intermediate F_v/F_m appeared in visibly normal tissue. Those became water-soaked approx. 24 h later and then turned necrotic. Later in the necrotrophic phase, there was a rapid increase in affected and necrotic tissue followed by a slower increase as necrotic areas merged. Treatment with the induced systemic resistance activator, 2R, 3R-butanediol, delayed affected and necrotic tissue development by approx. 24 h. Also, the halo of affected tissue was narrower indicating that plant cells retained a higher photosystem II efficiency longer prior to death. While chlorophyll fluorescence imaging can reveal much about the physiology of infected plants, this study demonstrates that it is also a practical tool for quantifying hemibiotrophic fungal infections, including affected tissue that is appears normal visually but is damaged by infection.     

Prochloraz for control of fungal pathogens of cultivated mushrooms

A wettable powder of prochloraz manganese complex 50% a.i. satisfactorily controlled the four major pathogens ofAgaricus bisporus andA. bitorquis: Verticillium fungicola var.fungicola, the cause of dry bubble,Mycogone perniciosa, the cause of wet bubble,Cladobotryum dendroides, the cause of cobweb disease andV. fungicola var.aleophilum, the cause of brown spots. The results were obtained in trials in trays with theAgaricus species, which were inoculated with the pathogens.The product was not toxic to bothAgaricus species in the effective dosages and the taste of the mushrooms was not affected by prochloraz formulations. Application of 1.5 g a.i. m^–2 nine days after casing is preferred, also in view of the residue levels. A 45% emulsifiable concentrate of the product without manganese was slightly toxic to mushrooms in a lower dosage than the wettable poweder.Samenvatting Een spuitpoeder met 50% prochloraz als werkzaam bestanddeel (een prochlorazmangaan-complex) gaf in de teelt van champignons (A. bisporus enA. bitorquis) een uitstekende bestrijding van de vier belangrijkste pathogene schimmels:Verticillium fungicola var.fungicola (de veroorzaker van droge mollen),Mycogone perniciosa (de veroorzaker van natte mollen),Cladobotryum dendroides (spinnewebschimmel) enV. fungicola var.aleophilum (de veroorzaker van bruine vlekken). Deze resultaten werden verkregen in proeven met kisten waarin deAgaricus-soorten, die geïnoculeerd werden met genoemde pathogenen, werden geteeld.Het produkt was voor geen van beideAgaricus-soorten toxisch als het werd toegepast in de werkzame doseringen, en de champignonsmaak werd door het middel niet nadelig beïnvloed. De voorkeur wordt gegeven aan toepassing van 1,5 g werkzame stof per m², 9 dagen na het afdekken, mede op grond van de gevonden residuwaarden. Een 45% vloeibare formulering zonder mangaan was, in lagere doseringen dan het spuitpoeder, in lichte mate toxisch voor champignons.     

Biocontrol options for Cirsium arvense with indigenous fungal pathogens

As a first step in the development of a mycoherbicide for biological control of Cirsium arvense indigenous fungal pathogens that had been isolated from diseased hosts were tested both singly and in combination on potted plants under environmental conditions. Disease symptoms (necroses, chloroses and macerations) and parameters of growth and development were evaluated to determine the potential for weed control. During 1998, Phoma destructiva, Phoma hedericola, a Mycelia sterilia and Puccinia punctiformis were inoculated singly. With the exception of the obligate biotroph P. punctiformis (local infections), the single inoculations reduced the measured parameters. A combined inoculum of P. punctiformis and Ph. hedericola was less effective than Ph. hedericola alone. In 1999, Ph. hedericola, Ph. destructiva, Ph. nebulosa and the Mycelia sterila were applied both singly and as a combined inoculum. Of all the tested inocula, the mixture of four pathogens had the greatest potential for biocontrol, significantly reducing reproductive capacity of the plants. These results demonstrate the importance of taking advantage of synergisms in developing a mycoherbicide.     

Research progress in the interactions of fungal pathogens and insect pests during host plant colonization

Journal of Plant Diseases and Protection - Microbial pathogens and insect pests frequently share the same host plant, and both can cause severe damage. When surviving on the same host, the fungal...     

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.     

A compartmental computer model of foliar uptake of pesticides

The uptake and movement of a chemical into a wheat leaf has been simulated in a 5-compartment model. The compound is considered to be applied to the leaf as discrete droplets of solution, from which water evaporates at a uniform rate. Solute diffuses from the droplets through the plant cuticle into the epidermal cell wall, and from there either into and out of the cytoplasm and vacuole of the mesophyll cells or along cell walls to the xylem. Once in the xylem it is carried in the transpiration stream towards the tip of the leaf. Accurate values for the parameters required by the model have not yet been measured, but the model simulates in a qualitative manner the movement pattern observed for 1-methyl-2-pyridone. The results presented focus attention on the possible importance of membrane permeability in determining the phytotoxicity of xylem mobile pesticides.     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

A new tool for entry and analysis of virulence data for plant pathogens

The methods of Habgood (1970) and Gilmour (1973) for the assignment of pathogen race names are discussed and the advantages of a reversed octal system, based on Gilmour's octal code, are emphasized. A new spreadsheet, 'HaGiS', exploits these two designation systems to provide user-friendly and fast access to some routine steps in the analysis of virulence data for plant pathogen populations. It produces graphs and tables for frequency distributions (of pathotypes, isolate complexities and cultivar susceptibilities); sorted lists (e.g. of the races ranked by predominance); indices (of sample diversity and of pathogenicity for each differential cultivar); and coloured proximity matrices. Use of HaGiS requires knowledge of only a few basic functions of MS Excel 97^®. All graphs, tables, indices and matrices are generated automatically and displayed by switching to the corresponding worksheet. In addition to data analysis, the spreadsheet is convenient for the entry of experimental data, allowing for all commonly used assessment scales.     

A cultivation independent,PCR-based protocol for the direct identification of plant pathogens in infected plant material

A protocol involving PCR, shot-gun cloning and sequencing was developed as a pre-diagnostic screening tool working directly on disease symptoms. The method was used to show the presence of biotrophic and non-biotrophic eukaryotic plant pathogens in leaves and fruits.     

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.     

Evolving strategies for biological control of weeds with plant pathogens

Current biological control strategies involve the use of exotic or native biocontrol agents, proven to be host-specific and lethal. This is a successful field of endeavor, attractive lo a large cadre of entomologists and a few plant pathologists. The main limitation is that each weed species must be attacked by a separate host-specific agent and these organisms are not always lethal. Only by using a radical paradigm shift can we reach a new strategy of biocontrol. In this strategy, lethal, broad host-range pathogens are genetically modified to permit their safe release. Either they are rendered host-specific or they are given a chemical dependency that prevents their spread or long-term survival. This genetic-manipulative approach offers numerous and diverse scenarios for biocontrol. Host-specific promoters or toxins, host-dependency by multiple auxotrophy, or mutants dependent on specific environmental conditions are all approaches that lend themselves to genetic modification. Such self-delimiting pathogens may have the advantage of repeated marketability, environmental safely and multiple target weeds.     

A new multiplex polymerase chain reaction assay for simultaneous detection of five soil-borne fungal pathogens in winter wheat

Journal of Plant Diseases and Protection - Wheat (Triticum spp.) is often coinfected by several soil-borne fungal pathogens, causing serious crop yield and economic losses. Rapid diagnosis of...     

An assessment model for rating high-threat crop pathogens

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.     

A nonlinear, dynamic, simulation model for transport, and whole plant allocation of systemic xenobiotics following foliar application. IV: Physicochemical properties requirements for optimum absorption and translocation

The relationship between the physicochemical properties (molar volume, partition coefficient, and dissociation constant) of slow-acting systemic postemergence xenobiotics and their uptake and translocation to the sites of action was investigated using the nonlinear, dynamic simulation model ERMESSE. When the pK_a was held constant at 4.0, the model enables the prediction of the uptake of a systemic xenobiotic as a function of its partition coefficient and molar volume. The model also considered the effects of the physicochemical properties of a systemic xenobiotic on its long-distance translocation within the vascular tissues. For instance, when the log K_ow and pK_a were held constant at 1.5 and 6.0, respectively, the model predicted a higher translocation rate (55%) for molecules with a small (e.g., MV = 100 cm³ mol⁻¹) as opposed to a large (e.g., MV = 300 cm³ mol⁻¹, 33%) molar volume. In addition, the theoretical predictions from the ERMESSE model showed that any xenobiotic with a molar volume not exceeding 300 cm³ mol⁻¹ could provide an uptake ?50% and a translocation rate ?25% when its log K_ow is between −0.5 and 2.5 and its pK_a is between 0.0 and 8.0.