In this study, the innovative development of two AmplifyRP® tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP® test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20 min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP® technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP® to detect PPV when compared to the conventional ELISA and ImmunoStrip® assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops.
Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real-time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue-printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real-time PCR. This simplified method was validated and could detect HLB-liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB-infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB-infected trees with or without symptoms. A commercial ready-made kit based on this technology showed 96% accuracy in intra-laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.
Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants).
Experts in Europe and Africa are racing to develop resistant grain varieties as university researchers predict the likely spread across continents of the air-borne spores of the fungus ---- Scientists are warning that wheat is facing a serious threat from a fungal disease that could wipe out the world’s crop if not quickly contained. Wheat rust, a devastating disease known as the “polio of agriculture”, has spread from Africa to South and Central Asia, the Middle East and Europe, with calamitous losses for the world’s second most important grain crop, after rice. There is mounting concern at the dangers posed to global food security.Experts have been aware of the threat since a major epidemic swept across North America’s wheat belt in the 1950s, destroying up to 40 per cent of the crop. Since then, tens of millions of pounds have been invested in developing rust-resistant varieties of the grain. However, an outbreak in Uganda in 1999 was discovered to have been caused by a virulent mutation of the fungus. There has been alarm at the speed at which further mutations have subsequently developed and spread across continents.Plant scientists in Britain estimate the latest developments mean that 90 per cent of all current African wheat varieties are now vulnerable to the disease.
Via Kamoun Lab @ TSL
A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3′-untranslated region (UTR) sequences of ToRSV, to amplify a 182 bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.
Pollination is an essential step in the reproduction of flowering plants and is also crucial in agriculture in regard to fruit development, seed output, and the creation of new varieties of plants. However, at least 18 viruses can infect the mother plant through the fertilized flower (horizontal transmission by pollen). Horizontal transmission by pollen is epidemiologically important for viruses infecting perennial crops, since pollen grains from infected trees continue to be scattered every year. The mechanism how pollination with virus-infected pollen grains causes systemic viral infection to healthy plants has been unknown since the first report of horizontal transmission by pollen in 1918.
IMPORTANCE Viruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrate that the RNA in one of the virions is released more easily than the other two in vitro. The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection.
The first droplet-digital-PCR-based absolute quantification of “flavescence dorée” phytoplasma, agent of a quarantine phytoplasma yellows in grapevine was performed. Quantitative PCR that targets the secY gene was transferred to droplet digital PCR and used for absolute quantification of “flavescence dorée” phytoplasma, without the need for calibration curves. The sensitivity of the two assays compares well and was shown that it could be used for quantification and quality control of DNA based on in-house reference materials typically used in diagnostics and metrological laboratories. This new tool has great potential for monitoring phytoplasma kinetics, such as there relationship with the progress of an infection, and variations of the phytoplasma titer through the season and screening plants for resistance.
Plant pathogens like ‘Ca. Liberibacter’, phytoplasma, viruses and viroids cause diseases to almost all economically important plants. A simple and fast sap-mediated polymerase chain reaction (PCR) method for the detection of these pathogens infecting various plant hosts is described in the present study. Sap from selected plants was drawn aseptically on parafilm, from the mid-rib of young leaves. Depending on the type of host plant, sap was diluted to optimal concentration before PCR analysis. ‘Ca. Liberibacter’, Citrus tristeza virus (CTV) and Citrus exocortis viroid (CEVd) infecting citrus, and ‘Ca. Phytoplasma’ infecting pepper and sandal trees were tested by sap-mediated PCR. The reliability of this procedure was evaluated by comparing the findings with previously described protocols. The sap-mediated nucleic acid template preparation for PCR assay is devoid of laborious nucleic acid extraction and expensive chemicals. Hence the present method is rapid, economical and so can be employed for diagnosis of large number of plant samples
Neurospora crassa has a long history as an excellent model for genetic, cellular, and biochemical research. Although this fungus is known as a saprotroph, it normally appears on burned vegetations or trees after forest fires. However, due to a lack of experimental evidence, the nature of its association with living plants remains enigmatic. Here we report that Scots pine (Pinus sylvestris) is a host plant for N. crassa. The endophytic lifestyle of N. crassa was found in its interaction with Scots pine. Moreover, the fungus can switch to a pathogenic state when its balanced interaction with the host is disrupted. Our data reveal previously unknown lifestyles of N. crassa, which are likely controlled by both environmental and host factors. Switching among the endophytic, pathogenic, and saprotrophic lifestyles confers upon fungi phenotypic plasticity in adapting to changing environments and drives the evolution of fungi and associated plants.
A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with ‘Candidatus Liberibacter solanacearum’ and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect ‘Ca. L. solanacearum’. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of ‘Ca. Liberibacter’ were observed using electron microscopy in celery plant tissues. A fifth haplotype of ‘Ca. L. solanacearum’, named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.
Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions.
The grass smuts comprise a speciose group of biotrophic plant parasites, so-called Ustilaginaceae, which are specifically adapted to hosts of sweet grasses, the Poaceae family. Mating takes a central role in their life cycle, as it initiates parasitism by a morphological and physiological transition from saprobic yeast cells to pathogenic filaments. As in other fungi, sexual identity is determined by specific genomic regions encoding allelic variants of a pheromone-receptor (PR) system and heterodimerising transcription factors. Both operate in a biphasic mating process that starts with PR–triggered recognition, directed growth of conjugation hyphae, and plasmogamy of compatible mating partners. So far, studies on the PR system of grass smuts revealed diverse interspecific compatibility and mating type determination. However, many questions concerning the specificity and evolutionary origin of the PR system remain unanswered. Combining comparative genetics and biological approaches, we report on the specificity of the PR system and its genetic diversity in 10 species spanning about 100 million years of mating type evolution. We show that three highly syntenic PR alleles are prevalent among members of the Ustilaginaceae, favouring a triallelic determination as the plesiomorphic characteristic of this group. Furthermore, the analysis of PR loci revealed increased genetic diversity of single PR locus genes compared to genes of flanking regions. Performing interspecies sex tests, we detected a high potential for hybridisation that is directly linked to pheromone signalling as known from intraspecies sex. Although the PR system seems to be optimised for intraspecific compatibility, the observed functional plasticity of the PR system increases the potential for interspecific sex, which might allow the hybrid-based genesis of newly combined host specificities.
Upon analysis of phytopathogen genomes it turned out that phytopathogenic microbes typically express dozens (bacteria; Collmer et al., 2009) to hundreds (oomycetes and fungi; Schmidt & Panstruga, 2011) of effector proteins. They often do not share any considerable sequence relatedness to known proteins and therefore can be considered as ‘pioneer proteins’, which renders their functional analysis a formidable task. Nevertheless, owing to the key role effector proteins play in plant–microbe interactions, their molecular analysis lately became very popular and is a flourishing research field. This development, which is evidenced by the substantial increase in literature devoted to ‘effectors’ during the last decade (Fig. 1), has also been appreciated by New Phytologist as documented by the organization of two symposia, in 2009 and 2012, with an emphasis on effectors in plant–microbe interactions (22nd New Phytologist Symposium and 30th New Phytologist Symposium; Lee et al., 2013). Besides proteinaceous effectors, secreted small molecules can also exhibit effector activity. Prominent examples from the phytopathogenic bacterium Pseudomonas syringae comprise syringolin (a proteasome inhibitor) and coronatine (a mimic of the phytohormone jasmonic acid), but also fungal secondary metabolites can have defense-suppressing activities (e.g. host-selective toxins; Tsuge et al., 2013).
In this Virtual Special Issue we compile a number of papers that were published recently in New Phytologist which all deal with various aspects of effector biology, ranging from bacterial to oomycete and fungal as well as nematode effectors. These papers cover effector functions related to suppression of plant immune responses as well as nutrient acquisition and the identification of plant effector targets.
Epichloё festucae is a filamentous fungus that forms a mutually beneficial symbiotic association with Lolium perenne. This endophyte synthesizes bioprotective lolitrems (ltm) and ergot alkaloids (eas) in planta but the mechanisms regulating expression of the corresponding subtelomeric gene clusters are not known. We show here that the status of histone H3 lysine 9 and lysine 27 trimethylation (H3K9me3/H3K27me3) at these alkaloid gene loci are critical determinants of transcriptional activity. Using ChIP-qPCR we found that levels of H3K9me3 and H3K27me3 were reduced at these loci in plant infected tissue compared to axenic culture. Deletion of E. festucae genes encoding the H3K9- (ClrD) or H3K27- (EzhB) methyltransferases led to derepression of ltm and eas gene expression under non-symbiotic culture conditions and a further enhancement of expression in the double deletion mutant. These changes in gene expression were matched by corresponding reductions in H3K9me3 and H3K27me3 marks. Both methyltransferases are also important for the symbiotic interaction between E. festucae and L. perenne. Our results show that the state of H3K9 and H3K27 trimethylation of E. festucae chromatin is an important regulatory layer controlling symbiosis-specific expression of alkaloid bioprotective metabolites and the ability of this symbiont to form a mutualistic interaction with its host.
Outbreaks of a deadly fungal disease in wheat crops in Germany and Ethiopia in 2013 have had the scientific community buzzing over the threat posed to global food security. Wheat stem rust, also known as wheat black rust, is often referred to as the “polio of agriculture”: The rapidly mutating fungal disease can travel thousands of kilometres and wipe out crops. Wheat farmers and scientists at a recent summit hosted by the Mexico-based International Maize and Wheat Improvement Center (CIMMYT ) have been examining outbreaks of different strains of wheat stem rust in the two countries to identify any similarities.
In Germany “the occurrence of stem rust was favoured by a period of unusually high temperatures… and an unusually late development of the wheat crop due to cold spring and early summer temperatures,” explained Kerstin Flath, senior scientist at Germany’s Federal Research Centre for Cultivated Plants at the Julius Kuehn-Institut. The outbreak occurred in June in central Germany, a mainly wheat producing area, and was the first in the country in several decades.
Then in November 2013 the disease struck a popular variety of wheat in Ethiopia called digalu, used to make bread, said Bekele Abeyo, a senior scientist and wheat breeder at CIMMYT. What was particularly disconcerting for the scientists was that digalu had been bred with inherent resistance to certain strains of stem rust and another wheat disease called “yellow rust” or “stripe”.
Genome architecture often reflects an organism’s lifestyle and can therefore provide insights into gene function, regulation, and adaptation. In several lineages of plant pathogenic fungi and oomycetes, characteristic repeat-rich and gene-sparse regions harbor pathogenicity-related genes such as effectors. In these pathogens, analysis of genome architecture has assisted the mining for novel candidate effector genes and investigations into patterns of gene regulation and evolution at the whole genome level. Here we describe a two-dimensional data binning method in R with a heatmap-style graphical output to facilitate analysis and visualization of whole genome architecture. The method is flexible, combining whole genome architecture heatmaps with scatter plots of the genomic environment of selected gene sets. This enables analysis of specific values associated with genes such as gene expression and sequence polymorphisms, according to genome architecture. This method enables the investigation of whole genome architecture and reveals local properties of genomic neighborhoods in a clear and concise manner.
The yam nematode, Scutellonema bradys, which can cause dry rot disease of yam (Dioscorea spp.), was recorded for the first time from Costa Rica in four species of yam occurring in the Atlantic and north regions. Morphometric measurements from two populations from each region using ten female and 11 male characters corresponded with previous descriptions of this species. Canonical discriminant analysis of the female morphometric data separated the populations by region, whereas no separation by region was evident using the male data. Analysis of DNA sequences from the ITS region indicated that populations from Costa Rica were monophyletic with S. bradys from West Africa and clearly distinct from other Scutellonema species. No genetic separation by geographic region or Dioscorea species host was observed between Costa Rica populations. Species-specific primers were developed from the ITS region and supported the identity of 17 populations from 15 locations in Costa Rica as S. bradys: 14 populations from D. alata (greater or water yam) and one each from D. trifida (white yampee), D. cayenensis (yellow yam) and D. rotundata (white yam). Yam production in Costa Rica began in the Atlantic region, where the yam nematode was likely introduced from the Caribbean, progressively spreading to other locations through the use of infected vegetative planting material.
Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing proteins (NB-LRRs or NLRs) to respond to invading pathogens and activate immune responses. How plant NB-LRR proteins respond to pathogens is poorly understood. We undertook a gain-of-function random mutagenesis screen of the potato NB-LRR immune receptor R3a to study how this protein responds to the effector protein AVR3a from the oomycete pathogen Phytophthora infestans. R3a response can be extended to the stealthy AVR3aEM isoform of the effector while retaining recognition of AVR3aKI. Each one of 8 single amino acid mutations is sufficient to expand the R3a response to AVR3aEM and other AVR3a variants. These mutations occur across the R3a protein, from the N-terminus to different regions of the LRR domain. Further characterization of these R3a mutants revealed that at least one of them was sensitized, exhibiting a stronger response than the wild-type R3a protein to AVR3aKI. Remarkably, the N336Y mutation, near the R3a nucleotide-binding pocket, conferred response to the effector protein PcAVR3a4 from the vegetable pathogen Phytophthora capsici. This work contributes to understanding how NB-LRR receptor specificity can be modulated. Together with knowledge of pathogen effector diversity, this strategy can be exploited to develop synthetic immune receptors.
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