Effective detection of pathogens from complex substrates is a challenging task. Molecular approaches such as real-time PCR can detect pathogens present even in low quantities. However, weak real-time ...PCR signals, as represented by high cycle threshold (C sub(t)) values, may be questionable. Therefore, setting a reliable C sub(t) threshold to declare a positive reaction is important for specific detection. In this study, five methods were assessed for their performance in determining a C sub(t) cut-off value. These methods were based on the widely used probability of detection (POD) or receiver-operating characteristic (ROC) approaches. Two important forest pathogens, Hymenoscyphus fraxineus and Fusarium circinatum, were used to set up three experimental frameworks that combined two types of substrates (seed lots and spore traps) and different PCR machines. The ROC-based method emerged as the most complete and flexible method under various experimental conditions. It was demonstrated that the ROC method leads to a cut-off value below which late C sub(t) results can reliably be considered indicative of positive test results. This cut-off value must be determined for each experimental approach used. The method based on the distribution of a previously determined set of C sub(t) values corresponding to false-positives appeared to be better adapted to detecting false-negative results, and thus useful for testing potentially invasive pathogens.
Gibberella circinata (anamorph Fusarium circinatum) is a harmful fungus infecting pine trees that has been subject to European Union emergency phytosanitary measures since 2007. This fungus is spread ...over long distances primarily via pine seeds, consequently reliable detection tools and methods are needed to test pine seed lots imported and marketed in the EU. Several detection methods are available in the scientific literature but very limited validation data is available. For that purpose, one European EUPHRESCO project called ‘Gibcir‐Diagseed’ was launched, involving 10 countries. This project aimed to select and validate one or more diagnostic procedures targeting this pest via inter‐laboratory tests, as well as a suitable sampling method. Performance criteria were calculated for three procedures chosen by participants: isolation followed by morphological characterization, isolation followed by conventional PCR and biological enrichment followed by real‐time PCR. The results showed that all three procedures provided acceptable performance criteria but also demonstrated that none of them could generate 100% of correct results. Regarding the sampling issue, the National Plant Protection Organizations consultation enabled an agreement to be reached on the number of seeds to be tested: 400 per sample, for a 95% confidence level that an infection of at least 1% will be detected.
Etude de performance des procédures de diagnostic pour l'identification et la détection de Gibberella circinata dans les semences de pin dans le cadre d'un projet EUPHRESCO
Gibberella circinata (anamorphe Fusarium circinatum) est un champignon pathogène infectant les pins qui fait l'objet de mesures phytosanitaires d'urgence au sein des pays de l'Union européenne depuis 2007. Ce champignon est disséminé sur des distances importantes essentiellement par le biais de semences de pin. Par conséquent, des outils et des méthodes de détection fiables sont nécessaires pour tester les lots de semences de pin importés et commercialisés dans l'Union européenne. Plusieurs méthodes de détection sont décrites dans la littérature scientifique mais il y a peu de données de validation disponibles. A cet effet, un projet européen EUPHRESCO nommé « Gibcir‐Diagseed » a été lancé en impliquant 10 pays. Ce projet vise à sélectionner et valider une ou plusieurs procédures ciblant cet organisme au travers de tests inter‐laboratoires, en même temps qu'une méthode d’échantillonnage adaptée. Des critères de performance ont été calculés pour trois procédures de diagnostic choisies par les participants: isolement suivi de la caractérisation morphologique, isolement suivi d'une PCR conventionnelle, et enrichissement biologique suivi par une PCR en temps réel. Les résultats ont montré que les trois procédures avaient des critères de performance acceptables mais ont également démontré qu'aucune d'entre elles ne pouvait générer 100% de résultats corrects. Au sujet de l’échantillonnage, la consultation des Organisations Nationales de la Protection des Végétaux a permis d'atteindre un consensus sur le nombre de semences à tester: 400 par échantillon, pour détecter un niveau d'infection d'au moins 1% avec un niveau de confiance de 95%.
Иccлeдoвaниe кaчecтвa тecтиpoвaния диaгнocтичecкиx пpoцeдyp пo идeнтификaции и выявлeнию Gibberella circinata в ceмeнax cocны в paмкax пpoeктa EUPHRESCO
Gibberella circinata (aнaмopфнaя cтaдия Fusarium circinatum) являeтcя вpeдным гpибoм, зapaжaющим cocнy, кoтopый являeтcя oбъeктoм чpeзвычaйныx фитocaнитapныx мep Eвpoпeйcкoгo Coюзa (EC) c 2007 г. Этoт гpиб pacпpocтpaняeтcя нa бoльшиe paccтoяния, пpeждe вceгo c ceмeнaми cocны, и пoэтoмy нeoбxoдимы нaдeжныe cpeдcтвa oбнapyжeния и мeтoды, пoзвoляющиe пpoвepять пapтии ceмян cocны, кoтopыe импopтиpyютcя и пpoдaютcя в EC. B нayчнoй литepaтype дaeтcя oпиcaниe нecкoлькиx мeтoдoв oбнapyжeния, oднaкo cyщecтвyют лишь oгpaничeнныe дaнныe o вaлидaции. C этoй цeлью был пpeдпpинят пpoeкт EC EUPHRESCO пoд нaзвaниeм “Gibcir‐Diagseed”, в кoтopый былo вoвлeчeнo 10 cтpaн. Этoт пpoeкт нaцeлeн нa oтбop и вaлидaцию oднoй или бoлee пpoцeдyp для идeнтификaции этoгo вpeднoгo opгaнизмa пyтeм тecтиpoвaния в нecкoлькиx лaбopaтopияx, a тaкжe пoдxoдящий мeтoд oтбopa oбpaзцoв. Кpитepии кaчecтвa aнaлизa были пoдcчитaны для тpex пpoцeдyp, выбpaнныx yчacтникaми: выдeлeниe c пocлeдyющeй xapaктepизaциeй мopфoлoгии, изoляция c пocлeдyющим oбычным ПЦP и биoлoгичecкoe oбoгaщeниe c пocлeдyющим ПЦP в peaльнoм вpeмeни. Peзyльтaты пoкaзaли, чтo вce тpи пpoцeдypы oбecпeчили пpиeмлeмыe кpитepии кaчecтвa, нo тaкжe пpoдeмoнcтpиpoвaли, чтo ни oдин из ниx нe мoг дaть 100%‐yю пpaвильнocть peзyльтaтoв. B oтнoшeнии oтбopa oбpaзцoв, кoнcyльтaция c нaциoнaльными opгaнизaциями пo кapaнтинy и зaщитe pacтeний пoзвoлилa бы пpийти к coглaшeнию пo кoличecтвy тecтиpyeмыx ceмян: 400 в кaждoм oбpaзцe, для 95%‐гo дoвepитeльнoгo ypoвня, пpи кoтopoм бyдeт выявлятьcя, пo кpaйнeй мepe, 1%‐нaя зapaжeннocть.
Filamentous fungi grow by the elaboration of hyphae, which may fuse to form a network as a colony develops. Fusion of hyphae can occur between genetically different individuals, provided they share a ...common allele at loci affecting somatic compatibility. Diversity in somatic compatibility phenotypes reduces the frequency of hyphal fusion in a population, thereby slowing the spread of deleterious genetic elements such as viruses and plasmids, which require direct cytoplasmic contact for transmission. Diverse somatic compatibility phenotypes can be generated by recombining alleles through sexual reproduction, but this mechanism may not fully account for the diversity found in nature. For example, multiple compatibility phenotypes of Fusarium circinatum were shown to be associated with the same clonal lineage, which implies they were derived by a mutation rather than recombination through sexual reproduction. Experimental tests of this hypothesis confirmed that spontaneous changes in somatic compatibility can occur at a frequency between 5 and 8 per million spores. Genomic analysis of F. circinatum strains with altered somatic compatibility revealed no consistent evidence of recombination and supported the hypothesis that a spontaneous mutation generated the observed phenotypic change. Genes known to be involved in somatic compatibility had no mutations, suggesting that mutation occurred in a gene with an as yet unexplored function in somatic compatibility.
•The RAS2 gene was successfully knocked out and replaced in the genome of in Fusarium circinatum.•RAS2 is important for growth in Fusarium circinatum.•RAS2 is essential for pathogenicity in Fusarium ...circinatum.•RAS2 plays a role in conidial germination in Fusarium circinatum.
In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.
Endophyte-host interactions lead to the production of various bioactive compounds. Thus, endophytes from a medicinal plant such as Momordica charantia, can be promising candidates for producing ...pharmacological compounds. Our study therefore aims to evaluate these endophytes as sustainable sources of potential low-cost lung cancer drugs. We determined the endophytes associated with M. charantia (fruit and leaf) and assessed anti-inflammatory, antioxidant and antiproliferative potential against NCI-H23 lung cancer cells. LC-Q-TOF-MS of the endophyte extracts was conducted to determine the metabolite profile.
Leaf endophyte F. circinatum showed anti-inflammatory (114.29 %), antioxidant (IC50=27.39 µg/ml) and antiproliferative activity (IC50=98.62 µl/ml) with decreased Beclin1 expression (autophagy regulator) in NCI-H23 cells. It produced five anticancer compounds namely linamarin, xestoaminol C, phytosphingosine, cucurbitacin E and margarolic acid. Fruit endophyte E. fergusonii also showed significant antiproliferative potential (IC50=170.8 µl/ml) with upregulated apoptosis regulator Bcl2 expression. It produced compounds such as eplerenone, kuguacin C and H. With the production of triterpenoids momordicine I, cucurbitacin E and kuguaglycoside A, leaf endophyte A. faecalis showed antioxidant potential (IC50=29.65 µg/ml) but limited antiproliferative activity. This is the first report of endophytes-mediated biosynthesis of host-specific compounds in M. charantia including momordicine I, cucurbitacin E and kuguacins, which are known to have anti-cancer properties. Thus, these medicinal plant endophytes can be low-cost sustainable candidates with anti-cancerous potential, especially against lung cancer.
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•Culturable endophytes density in Momordica charantia fruit was higher than leaf.•Promising antiproliferative and antioxidant activity by M. charantia endophytes.•Fusarium circinatum and Escherichia fergusonii promoted apoptosis in NCI-H23 cells.•M. charantia endophytes produced momordicine I, cucurbitacin E, kuguacin C and H.•Phytochemical biosynthesis varied with endophyte type and plant part interaction.
Pitch canker caused by the fungus
is an important disease affecting pine trees in Europe and South Africa. Several countries, including China, have listed
as a quarantine pathogen. Therefore, timely ...detection of
could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay was developed for rapid detection of
based on a new target gene,
, identified from whole-genome sequences. The assay was highly specific to
. In fact, it exclusively detected
isolates; 53 isolates of fungal and oomycete species and 2 nematodes of
and
were not detected. By detecting as little as 10 pg of
genomic DNA in a 50-µl reaction, the RPA-LFD assay was 10 times more sensitive than conventional PCR assays.
was also detected in artificially inoculated pine needles of
. These results demonstrated that the developed RPA-LFD assay has the potential for rapid detection of
in regions at high risk of infection. The RPA-LFD assay might serve as an alternative method for the early detection of
.
Coniferous trees can suffer significant damage from seedling diseases caused by Fusarium spp. and from pitch canker, which is caused by Fusarium circinatum Nirenberg & O'Donnell. These fungi can be ...responsible for both pre- and post-emergence damping-off, and latent infections can result in failure of transplanted seedling to become established. Fusarium oxysporum Schlechtend.: Fr. has long been recognized as the most important cause of root and hypocotyl rot in seedling nurseries, but phylogenetic analysis suggests that the most virulent strains are more properly assigned to the recently described Fusarium commune Skovgaard, O'Donnell et Nirenberg. Management of Fusarium diseases in bare-root seedling nurseries has relied primarily on preplant soil fumigation. Because regulations will increasingly limit availability of the most efficacious fumigants, alternative management practices are being explored. This includes greater attention to sanitation, maintaining a robust microbial community that will inhibit root-infecting pathogens and avoiding practices that predispose trees to disease, such as excessive fertilization and poorly drained soils. F. circinatum Nirenberg & O'Donnell can be a problem in seedling nurseries but is also damaging to mature trees in plantations, seed orchards, landscape plantings, and native forests. A critical element of management is limiting spread of disease from existing infestations. To this end, branches and logs removed from infected trees should be disposed of locally. Seeds and seedlings can both carry the pathogen and so should not be moved from infested to non-infested areas. Quarantine restrictions should be maintained to prevent introduction of F. circinatum into countries where it is not yet established. Infections caused by F. circinatum are associated with wounds resulting from silvicultural practices, such as pruning and seed harvesting, weather related injuries and insect activity. In managed plantings, the risk of disease can be reduced by limiting pruning operations to cool, dry periods, which are less conducive to infection, and by judicious control of insects that can serve as wounding agents and vectors. Variation in susceptibility to pitch canker has been documented in a number of commercially important pine species, offering the prospect for greater utilization of genetic resistance for management of this disease in the future.
•Alternatives to soil fumigation are needed for control of conifer seedling diseases.•Fusarium circinatum can be moved with seeds, seedlings and logs.•F. circinatum infects grasses, which may serve as inoculum reservoirs.•The impact of pitch canker is strongly influenced by predisposing stress on the host.•Induced resistance has slowed progress of pitch canker in Pinus radiata in California.
Fusarium circinatum is a pressing threat to the cultivation of many economically important pine tree species. Efforts to develop effective disease management strategies can be aided by investigating ...the molecular mechanisms involved in the host-pathogen interaction between F. circinatum and pine species. Pinus tecunumanii and Pinus patula are two closely related tropical pine species that differ widely in their resistance to F. circinatum challenge, being resistant and susceptible respectively, providing the potential for a useful pathosystem to investigate the molecular responses underlying resistance to F. circinatum. However, no genomic resources are available for P. tecunumanii. Pathogenesis-related proteins are classes of proteins that play important roles in plant-microbe interactions, e.g. chitinases; proteins that break down the major structural component of fungal cell walls. Generating a reference sequence for P. tecunumanii and characterizing pathogenesis related gene families in these two pine species is an important step towards unravelling the pine-F. circinatum interaction.
Eight reference based and 12 de novo assembled transcriptomes were produced, for juvenile shoot tissue from both species. EvidentialGene pipeline redundancy reduction, expression filtering, protein clustering and taxonomic filtering produced a 50 Mb shoot transcriptome consisting of 28,621 contigs for P. tecunumanii and a 72 Mb shoot transcriptome consisting of 52,735 contigs for P. patula. Predicted protein sequences encoded by the assembled transcriptomes were clustered with reference proteomes from 92 other species to identify pathogenesis related gene families in P. patula, P. tecunumanii and other pine species.
The P. tecunumanii transcriptome is the first gene catalogue for the species, representing an important resource for studying resistance to the pitch canker pathogen, F. circinatum. This study also constitutes, to our knowledge, the largest index of gymnosperm PR-genes to date.
The capacity to form biofilms is a common trait among many microorganisms present on Earth. In this study, we demonstrate for the first time that the fatal pine pitch canker agent, Fusarium ...circinatum, can lead a biofilm-like lifestyle with aggregated hyphal bundles wrapped in extracellular matrix (ECM). Our research shows F. circinatum's ability to adapt to environmental changes by assuming a biofilm-like lifestyle. This was demonstrated by varying metabolic activities exhibited by the biofilms in response to factors like temperature and pH. Further analysis revealed that while planktonic cells produced small amounts of ECM per unit of the biomass, heat- and azole-exposed biofilms produced significantly more ECM than nonexposed biofilms, further demonstrating the adaptability of F. circinatum to changing environments. The increased synthesis of ECM triggered by these abiotic factors highlights the link between ECM production in biofilm and resistance to abiotic stress. This suggests that ECM-mediated response may be one of the key survival strategies of F. circinatum biofilms in response to changing environments. Interestingly, azole exposure also led to biofilms that were resistant to DNase, which typically uncouples biofilms by penetrating the biofilm and degrading its extracellular DNA; we propose that DNases were likely hindered from reaching target cells by the ECM barricade. The interplay between antifungal treatment and DNase enzyme suggests a complex relationship between eDNA, ECM, and antifungal agents in F. circinatum biofilms. Therefore, our results show how a phytopathogen's sessile (biofilm) lifestyle could influence its response to the surrounding environment.
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