Diagnosis of plant diseases using the Nanopore sequencing platform

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single-molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.     

小麦矮腥黑穗病诊断技术研究进展

小麦矮腥黑穗病是由小麦矮腥黑穗病菌(Tilletia contraversa Kühn,TCK)引起的麦类腥黑穗病中危害最大,防治最难的一种病。该病是我国官方管制的检疫性植物真菌病害。本文从病菌冬孢子形态学诊断、生物学诊断、分子生物学诊断、图像识别诊断、以及电子鼻诊断5个方面综述了小麦矮腥黑穗病菌诊断技术,并对新诊断技术进行展望。     

New trends in orchard spraying1

Since the early part of the century, a steady move in England towards lower volume spraying of orchards has coincided with the progressive introduction of improved spraying techniques. In recent years many growers have adopted very low volume (VLV) spraying (50 litre ha^-1) using spinning-disc nozzles with greatly reduced dose rates, often only 25% of the product label recommendation. On well managed farms, this method of spraying has been successful for several years and considerable savings have been made in pesticide use and labour for spraying. On other farms there have been pest and disease control problems but few growers have reverted to spraying at conventional volume or pesticide rates. Trials evidence from the UK and the Netherlands shows the VLV reduced dose-rate spraying technique to be generally less effective than conventional spraying but usually sufficiently effective for commercial purposes. In the Netherlands a compromise method of low volume spraying (150–200 litre ha^-1) is being adopted on the recommendation of research and advisory services. This method of spraying is gaining popularity in England. Unfortunately, the method of application stipulated on product labels is usually restricted to a minimum water volume and a pesticide dose per ha. It might be desirable for experimental work for registration purposes to be done to explore optimum application methods for individual pesticides. This information on labels, in a simple form, might lead to the more efficient use of pesticides as well as reflecting the range of application methods in use in the industry.     

Insecticide resistance,control failure likelihood and the First Law of Geography

Insecticide resistance is a broadly recognized ecological backlash resulting from insecticide use and is widely reported among arthropod pest species with well‐recognized underlying mechanisms and consequences. Nonetheless, insecticide resistance is the subject of evolving conceptual views that introduces a different concept useful if recognized in its own right – the risk or likelihood of control failure. Here we suggest an experimental approach to assess the likelihood of control failure of an insecticide allowing for consistent decision‐making regarding management of insecticide resistance. We also challenge the current emphasis on limited spatial sampling of arthropod populations for resistance diagnosis in favor of comprehensive spatial sampling. This necessarily requires larger population sampling – aiming to use spatial analysis in area‐wide surveys – to recognize focal points of insecticide resistance and/or control failure that will better direct management efforts. The continuous geographical scale of such surveys will depend on the arthropod pest species, the pattern of insecticide use and many other potential factors. Regardless, distance dependence among sampling sites should still hold, following the maxim that the closer two things are, the more they resemble each other, which is the basis of Tobler's First Law of Geography. © 2016 Society of Chemical Industry     

Long-term cultivation and landscape position effects on aggregate size and organic carbon fractionation on surface soil properties in semi-arid region of Iran

This study was carried out to investigate the effects of long-term cultivation and landscape position on organic carbon content and soil aggregation. Sampling sites were determined based upon land use at the end of 50 years soil use and management, cultivated/annual wheat cropping and grazed pasture, and landscape position in Chaharmahal-va-Bakhtiary province, southwest Iran. Soil samples were collected from the 0–5 cm and 5–15 cm depths in two adjacent fields that have the same slope and aspect. The soil was silty clay at the summit and footslope positions, and was a silty clay loam at the backslope. Wet-sieving analysis and aggregate-size fractionation methods were used to separate the samples into three aggregate fractions (i.e., 2–4.75, 0.25–2, and 0.053–0.25 mm). The treatments were arranged in a factorial design. Land use significantly affected the water-stable aggregate fractions, so that the wet soil stability of the macroaggregates (i.e., 2–4.75 mm) was higher in the pasture, whereas it was greater for the meso-aggregates (i.e., 0.25–2 mm) in the cultivated soils. Cultivation decreased both the wet-aggregate stability and percent of macroaggregates whereas long-term pasture enhanced aggregation. Soil organic carbon (SOC) content within aggregates and primary particles was also significantly influenced by landscape position, land use, and the depth of sampling. The SOC content was higher in clay than those in silt and sand contents. The SOC content decreased as depth increased in all fractions. In general, the highest and lowest wet-stable aggregates were observed on the footslope and backslope positions, respectively.     

Development of an EU protocol for the detection and diagnosis of Potato spindle tuber pospiviroid*

The work described here formed part of the EU SMT DIAGPRO project, to develop diagnostic protocols for 18 regulated pests. The Potato spindle tuber pospiviroid (PSTVd) protocol was developed primarily for testing in vitro‐ and glasshouse‐grown potato plants for the purposes of post‐entry quarantine and the production of pathogen‐tested nuclear stock. After a performance audit of methods used by 12 laboratories in Europe and America by ring testing, four methods were chosen for multilaboratory validation. For most laboratories, the detection limits were 10–20 mg of PSTVd‐infective tissue for R‐PAGE; 0.25–0.5 mg for DIG‐probe; 0.062 mg for RT‐PCR; and 0.0155 mg for TaqMan (this was the lowest weight of infective tissue tested). Some laboratories were able to extend the detection limit to 0.0155 mg for DIG‐probe and RT‐PCR. The DIG‐probe and R‐PAGE are recommended as primary detection methods, with confirmation of viroid presence by any of the four validated detection methods. Specific diagnosis requires the viroid to be sequenced. Other methods may be used for primary detection, providing that they preferably detect all PSTVd isolates and other Pospiviroids that have the potential to infect potato, and detect viroid in at least 1/10 of the tissue weight normally tested per plant.     

Determinants of farmers’ decisions on pesticide use in oriental tobacco: a survey of common practices

Information about pesticide use and perceptions of their risks among farmers is important for identifying problems associated with pest-control decisions and developing appropriate management practices in given crops. In tobacco, a plant sensitive to many pests and diseases, such information is lacking. The patterns of pesticide use in oriental tobacco, as well as the most important factors for farmers’ decisions relating to chemical pest control were studied among randomly selected farmers in northern Greece on the basis of self-reported information acquired through a pre-tested questionnaire. High reliance of farmers on pesticides and field use of all well-known classes of chemical pesticides were reported. While all the active ingredients reported were registered for pest control in tobacco, they differed considerably in terms of toxicity. A normal cropping season received at least 14–16 pesticide applications for soil disinfection, pest control, and weed control. Most farmers stated that they usually spray chemicals on a calendar basis or on the first appearance of a pest. Efficacy on target pests and market price of the pesticide products to be used were the most important criteria for selection of pesticides, i.e. products that are considered to be highly effective and/or that are cheap are more likely to be selected. Better-educated and more experienced farmers were found to pay more attention to environmental safety and pesticide toxicity. Most farmers felt that pesticide use can reduce crop damage by more than 75%, assuring high yields. Main determinants of pesticide use were primarily the fear of severe yield and economic losses and the doubts about the effectiveness and the correct implementation of alternative pest-control methods. Pesticide use could be reduced by exposing farmers to improved pest-control practices such as site-specific integrated pest management.     

Diagnostic Values and Utility of Immunological, Morphological, and Molecular Methods for In Planta Detection of Phytophthora ramorum

ABSTRACT In this study, six methods for the detection of Phytophthora ramorum in planta were compared using naturally infested rhododendron plant material. The methods included two immunological methods, one an enzyme-linked immunosorbent assay (ELISA) and the other using a lateral flow format (LFD). Three molecular tests based on the polymerase chain reaction (PCR) using TaqMan chemistry also were assessed, including two assays designed for specific detection of P. ramorum and one designed for genus-level detection of Phytophthora. Isolation followed by morphological identification also was assessed. The diagnostic values of each of the methods, evaluated based on diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value, were calculated based upon the test results from 148 field samples. The "gold standard" used for the calculations was the final diagnosis, which was based on either a positive PCR result or successful isolation of P. ramorum. The Phytophthora spp. TaqMan PCR, ELISA, and LFD had higher sensitivities than the P. ramorum-specific methods, which make them useful as prescreening methods, where positive results must be confirmed by PCR or isolation. The article discusses practical advantages and disadvantages of each of the methods and how they are valuable in the diagnostic process, according to the circumstances of use (that is, diagnosis or surveillance) and in relation to the prevalence of P. ramorum infestation in the population to be tested.     

Molecular diagnostics for fungal plant pathogens

Accurate identification of fungal phytopathogens is essential for virtually all aspects of plant pathology, from fundamental research on the biology of pathogens to the control of the diseases they cause. Although molecular methods, such as polymerase chain reaction (PCR), are routinely used in the diagnosis of human diseases, they are not yet widely used to detect and identify plant pathogens. Here we review some of the diagnostic tools currently used for fungal plant pathogens and describe some novel applications. Technological advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Molecular methods have been used to detect several pathogens simultaneously in wheat, and to study the development of fungicide resistance in wheat pathogens. Information resulting from such work could be used to improve disease control by allowing more rational decisions to be made about the choice and use of fungicides and resistant cultivars. Molecular methods have also been applied to the study of variation in plant pathogen populations, for example detection of different mating types or virulence types. PCR-based methods can provide new tools to monitor the exposure of a crop to pathogen inoculum that are more reliable and faster than conventional methods. This information can be used to improve disease control decision making. The development and application of molecular diagnostic methods in the future is discussed and we expect that new developments will increase the adoption of these new technologies for the diagnosis and study of plant disease.     

The role of population and quantitative genetics and modern sequencing technologies to understand evolved herbicide resistance and weed fitness

Evolution of resistance to multiple herbicides with different sites of action and of nontarget site resistance (NTSR) often involves multiple genes. Thus, single‐gene analyses, typical in studies of target site resistance, are not sufficient for understanding the genetic architecture and dynamics of NTSR and multiple resistance. The genetics of weed adaptation to varied agricultural environments is also generally expected to be polygenic. Recent advances in whole‐genome sequencing as well as bioinformatic and statistical tools have made it possible to use population and quantitative genetics methods to expand our understanding of how resistance and other traits important for weed adaptation are genetically controlled at the individual and population levels, and to predict responses to selection pressure by herbicides and other environmental factors. The use of tools such as quantitative trait loci mapping, genome‐wide association studies, and genomic prediction will allow pest management scientists to better explain how pests adapt to control tools and how specific genotypes thrive and spread across agroecosystems and other human‐disturbed systems. The challenge will be to use this knowledge in developing integrated weed management systems that inhibit broad resistance to current and future weed‐control methods. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Detection and characterization of Plum pox virus: serological methods

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.     

Conventional tillage improves the storage of soil organic carbon in heavy fractions in the Loess Plateau,China

Soil labile organic carbon(C) plays an important role in improving soil quality. The relatively stable fractions of soil organic C(SOC) represent the bulk of SOC, and are also the primary determinant of the long-term C balance of terrestrial ecosystems. Different land use types can influence the distribution patterns of different SOC fractions. However, the underlying mechanisms are not well understood. In the present study, different fractions of SOC were determined in two land use types: a grazed grassland(established on previously cultivated cropland 25 years ago, GG) and a long-term cultivated millet cropland(MC). The results showed that C concentration and C storage of light fractions(LF) and heavy fractions(HF) presented different patterns along the soil profiles in the two sites. More plant residues in GG resulted in 91.9% higher LF storage at the 0–10 cm soil depth, further contributed to 21.9% higher SOC storage at this soil depth; SOC storage at 20–60 cm soil depth in MC was 98.8% higher than that in GG, which could be mainly attributed to the HF storage 104.5% higher than in GG. This might be caused by the long-term application of organic manure, as well as the protection from plough pan and silt- and clay-sized particles. The study indicated that different soil management practices in this region can greatly influence the variations of different SOC fractions, while the conventional tillage can greatly improve the storage of SOC by increasing heavy fractions.     

Effect of stale seedbed preparations and subsequent weed control in lettuce (cv. Iceboll) on weed densities

The effects of stale seedbed preparations and several weed control methods on the emergence of weeds in lettuce were studied. The specific goal was to evaluate the use of a stale seedbed in combination with chemical or mechanical weed control methods in the field. Depending on location and year, stale seedbed preparations followed by weed control prior to planting reduced the amount of weeds during crop growth by 43–83%. Control of the emerged seedlings after a stale seedbed preparation was more effective with glyphosate than with a rotary harrow. Covering the rotary harrow during control to prevent light reaching the soil improved its effect on the weed density during crop growth in two of 3 years. Radiation with far red light (FR) did not reduce the number of emerging weeds in this study. Mechanical control by finger weeder, torsion weeder and hoe was applied without stale seedbed preparations. These measures reduced the weed densities by 88–99%, compared with the untreated control and were more effective than chemical weed control with carbetamide and chlorpropham. The results show that the stale seedbed technique in combination with mechanical control of emerging weeds can reduce the weed population during crop growth as effectively as chemical control. The technique may therefore help reduce the use of herbicides in lettuce crops in the future.     

Nature and importance of weeds in sorghum in the Yemen Arab Republic

Abstract

Grain sorghum (Sorghum bicolor (L.) Moench) is the dominant crop in North Yemen agriculture, covering about 70–80% of the arable land. Weed control is still carried out mainly by hand. Many of the weeds are used as fodder for animals. A total of 137 weeds was collected in sorghum. Most frequent were Cynodon dactylon (L.) Pers. and Cyperus mtundus (L): their importance decreased, however, under a well‐established sorghum crop providing good shade cover and under conditions of increased crop fertilization with mineral nitrogen. Under these conditions, for example, Commelina spp. and Flaveria trinerva (Spreng.) Mohr became commoner. Thus increased use of nitrogen altered the nature and hierarchy of the weed flora. Relationships between the indicator figures of the weeds and the habitat, as defined by Ellenberg, were confirmed. Traditional methods of weed control have so far worked well, as witness an average cover of 60% sorghum and 20% weeds. In future, however, rising costs and labour shortages will increase the demand for less time‐consuming weed control strategies.     

Insecticide resistance in Musca domestica L. from Eastern England

Strains of houseflies were obtained from a refuse tip and 34 farms selected at random from Eastern England in 1984. Resistance was assessed to four insecticides that had been in use for fly control in the UK. Dose-response data were obtained by topical application of the insecticides and, in addition, a resistance test method was used in which flies were allowed to feed on sugar coated with either methomyl or azamethiphos. For both methods, the knock-down (KD) after 48 h was used as a measure of response. The ranges of resistance factors for the topical application method were, for methomyl, 1·1–15·9 at KD₅₀ and 2·1–17·0 at KD₉₅; for azamethiphos, 2·2–36·9 at KD₅₀ and 4·5–88·2 at KD₉₅; for pyrethrins + piperonyl butoxide, 0·9–6·8 at KD₅₀ and 1·4–13·4 at KD₉₅; and for permethrin, 1·7–34·8 at KD₅₀ and 2·2–112·5 at KD₉₅. The ranges of the resistance factors for the feeding tests were, for methomyl, 2·1–39·8 at KD₅₀ and 2·4–64·0 at KD₉₅, and, for azamethiphos, 5·6–51·7 at KD₅₀ and 8·8–92·0 at KD₉₅.     

The impact of land use change on soil organic carbon and labile organic carbon stocks in the Longzhong region of Loess Plateau

Land use change (LUC) is widely recognized as one of the most important driving forces of global carbon cycles. The soil organic carbon (SOC) and labile organic carbon (LOC) stores were investigated at arable land (AL), artificial grassland (AG), artificial woodland (AW), abandoned arable land (AAL) and desert steppe (DS) in the Longzhong region of the Loess Plateau in Northwest China. The results showed that conversions from DS to AL, AL to AG and AL to AAL led to an increase in SOC content, while the conversion from DS to AW led to a decline. The differences in SOC content were significant between DS and AW at the 20-40 cm depth and between AL and AG at the 0-10 cm depth. The SOC stock in DS at the 0-100 cm depth was 39.4 t/hm 2 , increased by 28.48% after cultivation and decreased by 19.12% after conversion to AW. The SOC stocks increased by 2.11% from AL to AG and 5.10% from AL to AAL. The LOC stocks changed by a larger magnitude than the SOC stocks, which suggests that it is a more sensitive index of carbon dynamics under a short-term LUC. The LOC stocks increased at 0-20 cm and 0-100 cm depths from DS to AW, which is opposite to that observed for SOC. The proportion of LOC to SOC ranged from 0.14 to 0.20 at the 0-20 cm depth for all the five land use types, indicating low SOC dynamics. The allocation proportion of LOC increased for four types of LUC conversion, and the change in magnitude was largest for DS to AW (40.91%). The afforestation, abandonment and forage planting on arable land led to sequestration of SOC; the carbon was lost initially after afforestation. However, the carbon sink effect after abandonment may not be sustainable in the study area.     

Image analysis for the identification of the quarantine pest Tilletia indica*

Diagnosis of the teliospores of Tilletia indica (Karnal bunt) detected in wash tests is complicated by the fact that wheat grain can be contaminated by ‘look‐alike’ species, namely Tilletia walkeri (ryegrass bunt) and Tilletia horrida (rice smut). Although morphological diagnosis is possible when there are relatively large numbers of teliospores present, it is difficult with only a few spores as there are significant overlaps in the characteristics of each species. Molecular methods can confirm presumptive identifications, but these take 2–3 weeks. Image analysis offers the potential for more rapid confirmation. An image analysis system was developed for use on bleached spores. Bleaching spores reveals additional morphological characters (spore profile and spore wall layers) that may be used in an image analysis system to discriminate species. The image‐processing software automatically locates spores on a given image and calculates perimeter, surface area, number of spines and spine size, maximum and minimum ray radius, aspect ratio and roundness. Principal components analysis (PCA) is performed on the parameters to obtain a linear separation of spore species. Accuracy of 97% in separating T. indica and T. walkeri has been achieved in preliminary tests using PCA, but further evaluation is required.     

Quantitative and qualitative influence of inoculation methods on in planta growth of rice blast fungus

ABSTRACT Molecular analyses of early disease events require infected plant tissue in which the pathogen is present in high quantities and interacts with the plant in a way found in the field. In this study, a quantitative polymerase chain reaction (Q-PCR) assay was developed to determine an "infection ratio" of fungal to plant cells in infected tissues. This assay was used to evaluate four inoculation methods (spray, mist, dip, and sheath) as well as use of whole plants or excised parts. Fluorescence stereomicroscopy was used to follow individual lesions developing from appressoria to macroscopic symptoms. Disease progression and outcomes were documented from 24 to 96 h postinoculation (hpi), as well as effectiveness of Pi-ta-mediated resistance. Even at 96 hpi, fungus proliferated well ahead of visible plant damage, especially in veins. Developing lesions sometimes were surrounded by greener areas in detached leaves. Spray inoculation was not sufficient for detecting fungal gene expression in planta before 96 h. Alternatively, a leaf sheath assay produced infected tissues containing 10 to 30% fungal DNA by 34 h. Used together, Q-PCR quantification and fluorescence stereomicroscopy will facilitate studies of early plant invasion because infection density and fungal growth stages are directly observed, not assumed from incubation time.     

Instrumental methods for the analysis of herbicide residues*

Instrumental techniques for the residue analysis of herbicides in soil, water and plant material are reviewed. Various analytical methods, including gas chromatography using different detector systems, thin-layer chromatography, colorimetry, spectroscopy, as well as the use of labelled compounds for the evaluation of residue methods, are discussed.