Rapid detection of Fusarium circinatum propagules on trapped pine beetles

Pitch canker is a destructive disease of pine caused by the fungus Fusarium circinatum. This taxon is listed as a quarantine fungus for several regional plant protection organizations throughout the world. Whereas long‐distance spread of the disease is made possible through the trade of infected pine seeds, local spread is caused by aerial dispersion or insect transportation of the fungal conidia. Developing a reliable and efficient tool to detect of F. circinatum in insects would be very useful to monitor the local spread of the pathogen. This tool would also provide the means to assess the range of insect species that could serve as potential vector of the fungus. A DNA extraction protocol was optimized and combined with a real‐time PCR test to detect F. circinatum on pine beetles. Using artificially contaminated Ips sexdentatus, it was shown that the test was able to detect down to 10 F. circinatum conidia per individual, and 20 conidia per batch of 10 insects, which is below the lowest inoculum load occurring in nature. With this technique, several batches of up to 10 insects may be analysed simultaneously, with a timescale for analysis reduced to <5 h and without the need for expertise in Fusarium taxonomy. This tool may be useful to monitor potential spread of the pathogen across regions. Using this method, to date, despite F. circinatum foci occurred in Northern Spanish regions across the border in France, the pathogen was not found on I. sexdentatus.     

Evaluation of Pinus radiata seed treatments to control Fusarium circinatum: effects on seed emergence and disease incidence

In this study, the effects of hot water (HWT), hydrogen peroxide and fungicides on the incidence of Fusarium circinatum on artificially inoculated Pinus radiata seeds were evaluated. Fifteen commercial fungicide formulations were screened in vitro for inhibitory activity on mycelial growth and conidial germination of F. circinatum. With half‐maximal effective concentration (EC₅₀) lower than 0.5 ppm, fluazinam, imazalil and tebuconazole were the most effective fungicides on mycelial growth, while captan, mancozeb or pyraclostrobin were the most effective (EC₅₀ < 0.3 ppm) on conidial germination. Based on the results obtained, imazalil, fluazinam, mancozeb and pyraclostrobin were selected for further testing. The effects of HWT, hydrogen peroxide and fungicide treatments on seed emergence and the incidence of F. circinatum were assessed. Seed treatments with fungicides prior to sowing were less effective and inconsistent in reducing the incidence of F. circinatum on seedlings. In contrast, hot water and hydrogen peroxide treatments significantly reduced F. circinatum contamination on P. radiata seeds with an overall disease incidence lower than 0.8% on seedlings. Furthermore, subsequent application of fungicides on seedlings did not improve the effectiveness of HWT. These results, therefore, suggest that hot water is a better alternative to hydrogen peroxide and fungicides as Pinus seed treatment against F. circinatum and could easily be implemented as standard in commercial nurseries to control the spread of the pitch canker disease.     

Fraxinus excelsior seed is not a probable introduction pathway for Hymenoscyphus fraxineus

We investigated the transmission of Hymenoscyphus fraxineus from infested seed to germinating seedlings of common ash (Fraxinus excelsior) in order to determine the potential risk associated with intra‐ and intercontinental movement of seed. Neither fungal isolations from necrotic or healthy embryos nor PCR testing with H. fraxineus‐specific primers detected the pathogen. Similarly, H. fraxineus was not detected in axenically grown seedlings generated from infested seed lots. The results help clear up prior confusion of the pathogen being seed‐borne. Any remaining surface contamination by pathogen spores could be washed off seeds as a quarantine measure.     

Exposure to a pine pathogen enhances growth and disease resistance in Pinus radiata seedlings

Most studies of Fusarium circinatum, the cause of pitch canker in pines, have focused on its activity as a pathogen. However, recent findings indicate that this fungus can colonize roots of Pinus radiata without inducing symptoms. Contrary to expectations, this study revealed that seedlings grown in infested sand grew more rapidly than seedlings not exposed to F. circinatum, based on root and shoot biomass, with modifications to root system architecture, including increased mycorrhizal root development. These effects were dependent on inoculum density and duration following growth in infested rooting medium. Plants exposed to F. circinatum expressed elevated resistance to stem infections, which significantly decreased the incidence of mortality; as above, effects were dependent on inoculum density. Resistance to stem infections was also enhanced in seedlings that emerged through infested litter, as occurs in native stands. Beneficial to neutral interactions of F. circinatum with its host suggest that the life history of this fungus may be more complex than previously recognized, with activities similar to non‐pathogenic endophytes. The potential for non‐lethal infections by F. circinatum to induce resistance in seedlings may influence dynamics of stand establishment. Overall, these results indicate that pathogenic organisms with asymptomatic states may have cryptic ecological functions that extend beyond the impacts of disease.     

Pitch canker ratings of longleaf pine clones correlate with Fusarium circinatum infestation of seeds and seedling mortality in containers

The incidence and severity of pitch canker was rated among 16 clones of mature longleaf pine (Pinus palustris) in a seed orchard and cones were collected from five of those clones across the range of pitch canker disease ratings. Seed were extracted and commercially processed by clone and the percentages contaminated with Fusarium circinatum determined. Seed from each clone were sown either without treatment or with one of three fungicide treatments in a soil‐less mix at a commercial container nursery to evaluate the effects of F. circinatum (syn. F. subglutinans) on seed and seedling survival. The percent of seed with F. circinatum correlated with the pitch canker ratings in the orchard for the year of collection (r = 0.88, p = 0.05) and, when sown without fungicide treatment, with the number of seedlings produced (r = ?0.94, p = 0.01) and with seedling mortality after germination (r = 0.92, p = 0.02). The same orchard clones were more symptomatic of pitch canker through three annual surveys. Fusarium circinatum was isolated from a higher percent of seed from more symptomatic clones and a lower percent of their seed produced plantable seedlings. We propose that removing such clones from seed collections should reduce seedling mortality at the nursery.     

Testing of selected South African <Emphasis Type="Italic">Pinus</Emphasis> hybrids and families for tolerance to the pitch canker pathogen, <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

Plantations of Pinus spp. constitute approximately 50% of the South African forestry industry. The first aim of this study was to develop a reliable inoculation technique to screen Pinus spp., for tolerance to infection by F. circinatum, which threatens pine forestry in South Africa. Inoculation of branches was compared with stem inoculations and we considered the number of branches or trees required to obtain statistically significant results. Furthermore, variation in the susceptibility of some Pinus families, clones and hybrids was considered. Results showed that branch inoculations were closely correlated with those from stem inoculations, and that it is important to consider branch and stem diameters when assessing susceptibility of trees. Subsequent trials using branch inoculations showed significant differences in F. circinatum tolerance amongst a range of pine species and hybrids of potential interest to forestry in South Africa. Significant differences in susceptibility were also found among clones of two P. radiata families. The most tolerant trees were P. elliottii × caribaea and P. patula × oocarpa hybrids, while the most susceptible species were P. patula, P. greggii and hybrids of these two. This is the first trial considering the susceptibility of Pinus hybrids, Pinus clones and some P. patula provenances, and the results indicate excellent potential for breeding for tolerance to pitch canker in South Africa. Application The accurate selection of disease tolerant planting stock for the South African forestry industry is crucially important for the continued sustainability of this important industry. The work described here provides valuable information on an artificial inoculation technique that will assist the industry in screening trees for tolerance to the pitch canker fungus, F. circinatum. It also provides some indication of the relative susceptibility of a number of Pinus spp., hybrids and families currently being evaluated in the country.     

Assessment of molecular detection of Fusarium circinatum in insects and passive spore traps in Pinus radiata plantations

Fusarium circinatum is the causal agent of pitch canker, a destructive disease that threatens natural and planted pine forests around the world. Although pitch canker has caused problems in Spain and Portugal, concerning Europe as a whole, the fungus is not established across the pine distribution area. Its dispersion by wind and/or insect vectors could nevertheless play a role in the colonization of currently uninfected stands. It is therefore crucial to develop monitoring tools for its detection. To this end, we assessed the molecular detection of the pathogen in environmental samples of bark beetles and passive spore traps, collected in two infected Pinus radiata plantations in Basque country, Spain. The spread pattern of F. circinatum was assessed by an experimental design that included insect and spore traps installed at the centre, at the edge and outside the plots. Our results showed that F. circinatum was detected in both types of samples, at almost all collection dates. In both type of samples, positive detections were mainly found at the centre of the plots, a lower proportion at the edge, and very few outside. This suggests that long‐distance dispersion of Fusarium circinatum does not rely on wind spore dispersal neither on insect flight. Our study also shows that molecular methods are a powerful tool to monitor the pathogen in environmental samples.     

Tolerance of Pinus patula full-sib families to Fusarium circinatum in a greenhouse study

The pitch canker fungus, Fusarium circinatum, has caused large-scale mortality of young Pinus patula Schiede and Deppe ex Schltdl. and Cham. seedlings in nurseries in South Africa since 1990. Diseased seedlings have been inadvertently carried to the field, which in turn have died and has reduced stocking below an acceptable level. Tree breeders have suggested that the only long-term solution to limit infection by this pathogen is to identify and deploy tolerant P. patula families. A commonly used technique to identify tolerant clones is to artificially inoculate open-pollinated progeny from orchard clones with F. circinatum under greenhouse conditions. In these trials, large variation in tolerance to the pathogen among seedlings within open-pollinated families was observed and this could be influenced by the pollen parent. Therefore, identifying individual full-sib families, where both parents are known, should improve the identification of tolerant families, which can then be repeated. In this study, cuttings from control-pollinated P. patula seedling hedges were inoculated with F. circinatum in a greenhouse. The results showed large family variation where some of the full-sib families were similar in tolerance to P. elliottii Engelm. var. elliottii seedlings. Therefore, it is recommended that breeders focus on identifying specific family combinations that are more tolerant to F. circinatum.     

Some physical conditions for removal of mechanically damaged pinus sylvestris L. seeds by using the PREVAC method

If low absolute pressure is first applied to and then released from seeds of Pinus sylvestris L. lying in water, the seeds with mechanical damage, i.e. with seed coat cracked, with part of seed coat missing or with part of seed coat and gametophyte missing, quickly lose their buoyancy and sink and can thus be separated from the floating undamaged seeds (PREVAC method). The most crucial of the tested physical properties was the level of pressure. The time of treatment (1–20 min) was of less importance, and the water temperature (5–40°C) was unimportant. Treatment at approximately 97 kPa below atmospheric pressure (3–5 kPa absolute pressure) for 1 up to 20 min resulted in removal of almost all mechanically damaged seeds in the four studied seed lots (containing up to 26% damaged seeds). In total, less than 5% undamaged seeds of the whole seed lots were removed. The germination percentage of the fractions remaining after separation increased with increasing number of damaged seeds in the seed lots and vice versa for the removed fractions.     

Individual resistance of Fraxinus angustifolia clones to ash dieback

The presumed resistance of individual ash trees to ash dieback caused by invasive pathogen Hymenoscyphus fraxineus is an important issue for the maintenance of ash in European forests. All known studies regarding the resistance of ash trees to ash dieback were conducted in plantations and stands of F. excelsior; however, no such data exist for F. angustifolia. Crown damage assessments were performed over four consecutive years between 2009 and 2012 at a F. angustifolia clonal plantation in Hra??ica, Slovenia. Inoculation of H. fraxineus into the branches of the most and least damaged clones of F. angustifolia and leaf phenology assessments was performed to verify the presence of defence mechanisms that limit fungal growth or promote disease escape. Additionally, root collars of selected clones were inspected for fungal infections. The crown damage assessments showed considerable differences among F. angustifolia clones, indicating genetic variability in susceptibility to ash dieback. Crown dieback progressed significantly over the 4‐year time period; the mean crown damage of individual clones in 2012 varied between 16.7% and 83.8%. Significant differences among F. angustifolia clones were found in the inoculation trials and leaf phenology assessments. However, defence mechanisms such as early leaf flushing, early leaf shedding and the ability to inhibit pathogen growth in host tissues were not confirmed. High frequency of Armillaria spp. and H. fraxineus root collar infection demonstrated the need for whole tree inspection to determine causal agent of damages on individual ash trees. Armillaria spp. may be highly associated with ash decline epidemiology.     

A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers

Ten taxon‐specific primers were designed to amplify the Internal Transcribed Spacer of the rRNA operon of several important decay fungi of coniferous wood, including Armillaria spp., Echinodontium spp., Fomitopsis pinicola, Fuscoporia torulosa, Heterobasidion annosum sensu lato (s.l.), Onnia spp., Phaeolus schweinitzii, Phellinus weirii s.l., Pholiota spp. and Porodaedalea spp. Primers designed in this study and in a previous one for the identification of Laetiporus sulphureus and Stereum spp. were combined in two multiplex PCRs, which were tested for efficiency and specificity, and detected at least 1 pg of fungal target DNA. Target DNA at concentrations of 10^?1 pg or lower can be detected with this assay using SYBR^® Green Real‐Time PCR. Validation assays performed on 129 naturally infected wood samples or fruiting bodies confirmed the reliability of the multiplex PCR‐based diagnostic method. This method represents a simple and rapid diagnostic tool for the detection of a number of destructive wood decay fungi of conifer wood.     

High genetic diversity of Fusarium circinatum associated with the first outbreak of pitch canker on Pinus patula in South Africa

The disease known as pitch canker results from infection of Pinus species by the fungus Fusarium circinatum. This fungus also causes a serious root disease of Pinus seedlings and cuttings in forestry nurseries. Pinus radiata and P. patula are especially susceptible to the pathogen, but there are no records of pitch canker on P. patula in established plantations. To date, only planting material of this tree species in nurseries or in plantations at the time of establishment have been infected by F. circinatum. Symptoms of pitch canker have recently emerged in an established P. patula plantation in South Africa and this study sought to determine whether the symptoms were caused by F. circinatum. Isolates from cankers were identified as F. circinatum using morphology and DNA-based diagnostic markers. Microsatellite markers were then used to determine the genetic diversity of a collection of 52 isolates. The entire population included 17 genotypes representing 30 alleles, with a greater number of genotypes collected from younger (three- to six-year-old) than older (12- to 19-year-old) trees. Both mating types of F. circinatum were present, but no evidence of sexual recombination was inferred from population genetic analyses. This is the first record globally of pitch canker on P. patula trees in managed plantations. It is of significant concern to South Africa, where P. patula is the most important Pinus species utilised for plantation forestry.     

Seedborne fungi in Norway spruce: testing methods and pathogen control by seed dressing

Italian seed lots of Norway spruce (Picea abies) were analysed by agar plate tests and seedling symptom test. Fusarium spp., Phoma spp. and Sirococcus conigenus were present on seeds, together with some saprophytes, but only S. conigenus and Fusarium spp. caused mortality in the seedling symptom test. Pathogenicity tests with 19 isolates belonging to nine Fusarium species showed that only some were pathogenic. Fusarium dlamini, reported for the first time in Italy, caused only some pre-emergence mortality. Pathogenicity tests with S. conigenus indicated that the isolate was pathogenic and that temperature can greatly affect the results. Seed dressing with prochloraz was suitable for controlling the seedborne pathogens. Agar plate tests are effective and practicable methods for the detection of spruce seed mycoflora.     

Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real‐time PCR

A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.     

Development of a quantitative real‐time PCR assay for the detection of Phytophthora austrocedrae,an emerging pathogen in Britain

A TaqMan real‐time PCR assay was developed for Phytophthora austrocedrae, an emerging pathogen causing severe damage to juniper in Britain. The primers amplified DNA of the target pathogen down to 1 pg of extracted DNA, in both the presence and absence of host DNA, but did not amplify any of the non‐target Phytophthora and fungal species tested. The assay provides a useful tool for screening juniper populations for the disease.     

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Abstract

The effect of soaking on germination and occurrence of fungal infections on Norway spruce (Picea abies (L.) Karst.) seeds and germinants was studied in three commercial seed lots. Treatments in which seeds were soaked in water for 24 h or in which the water was changed during the soak did not have an effect on the species diversity or on the abundance of the fungi isolated from the seeds. Different fungi were found in different seed lots. Most of those isolated are saprophytic or weakly pathogenic, but pathogens such as Sirococcus conigenus (D.C.) P.F. Cannon &; Minter and Trichothecium roseum (Pers.) Link were also isolated. Soaking increased germination energy but had no effect on final germination percentage. The number of mouldy seeds and germinants with disease symptoms was different among seed lots when seeds were germinated on water agar. When germinated in low humified Sphagnum peat, no differences in the emergence of disease symptoms were observed among seed lots. The frequency of disease was lower in peat than in water agar. Soaking had no effect on disease emergence in germination trials on either water agar or in Sphagnum peat.     

Responses of conifer species of the Great Lakes region of North America to inoculation with the pitch canker pathogen Fusarium circinatum

Five conifer species grown in the Great Lakes region of North America were examined for their susceptibility to Fusarium circinatum, (syns. Fusarium subglutinans f. sp. pini and F. moniliforme var. subglutinans), the causal agent of pitch canker. Three‐year‐old (3‐0) seedlings of red (Pinus resinosa), jack (P. banksiana) eastern white (P. strobus), Scots (P. sylvestris) and Austrian (P. nigra) pine were planted in 4 l pots in a greenhouse at Auburn University in November 1998. In April and June 1999, seedlings were inoculated by removing a needle fascicle approximately 5 cm from the terminal bud and placing a drop containing F. circinatum conidia on the wound. Resin production, canker length and seedling mortality were recorded 12 weeks later. Jack, Scots and eastern white pine were the most susceptible with Austrian and red pine more resistant to the fungus. F. circinatum was re‐isolated from 37% to 96% of inoculated seedlings. The susceptibility of jack, Scots and eastern white pine indicates a potential risk to these important species of the region if F. circinatum were to be introduced into the area.     

Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR

A real time PCR assay was developed for the detection of Chalara fraxinea in common ash. PCR primers and Taqman probes, based on the internal transcribed spacer region of the multi‐copy gene rDNA, were tested for specificity and sensitivity. The primers amplified an 81 bp fragment for C. fraxinea but did not amplify DNA from other Chalara species or from other fungi isolated from ash, whether pathogenic or saprophytic. The limit of detection was 5 pg of genomic DNA per PCR. Moreover, naturally‐infected samples were correctly diagnosed. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of an appropriate quality for real time PCR. This molecular method could be useful for routine analysis of this emergent pathogen and for epidemiological studies.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

A new method to detect Lonsdalea quercina in infected plant tissues by real‐time PCR

Presymptomatic and accurate diagnoses of pathogens are essential for disease prediction and the timely application of bactericide. The bacterium Lonsdalea quercina (=Brenneria quercina) has been reported as the causal agent of drippy nut and bark canker disease on oak in California (US) and Europe. In recent years, it is also found on Populus × euramericana trees in Henan province of China. This bacterium causes longitudinal cankers of a few centimetres in size on the bark surface of the upper trunk. In this study, we developed two species‐specific PCR assays using primer pairs LqfF/LqfR and LqgF/LqgR for the rapid and accurate detection of the pathogenic bacteria in diseased plant tissues. The results show that the LqfF/LqfR primers amplified only a single PCR band of approximately 382 bp and the LqgF/LqgR primers yielded a PCR product of approximately 286 bp. The two primers were successfully adapted to real‐time PCR based on SYBR Green I used with the ABI 7500 system. The detection limit of the reaction was 0.1 pg genomic DNA per 20 μl PCR reaction volumes. The pathogen was mainly detected in the phloem of cankers as well as in the exudates of diseased trees, but was not found in the xylem or leaves. The size of pathogen in distribution was larger than the lesion. The results demonstrate that real‐time PCR assays can be used to detect the pathogen by extracting DNA directly from infected plant tissues. This method is a rapid, reliable method for the presymptomatic and accurate detection of L. quercina, providing a useful insight into epidemiological studies.