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Pathogen life history traits influence epidemic development and pathogen adaptive ability to interact with their hosts in different environments. Reduced traits variation may compromise pathogen evolutionary potential, which is particularly important for introduced pathogens. Fusarium circinatum (cause of pine pitch canker) is an invasive fungal pathogen in Europe, with current distribution restricted to forest stands of Pinus radiata and Pinus pinaster in northern Spain and Portugal. This study aimed to quantify pathogenic traits of Spanish isolates of F. circinatum, with two of the strains representing the two dominant haplotypes in the Spanish population. Disease severity was measured on P. radiata, analyzing the influence of temperature and moisture duration on infection as well as the influence of temperature on spore germination, sporulation, and mycelial growth. Results indicated that the isolate representing the most common haplotype caused more severe disease on P. radiata at 25 and 30°C compared with the second most common haplotype but caused less severe disease at 15°C. Spore germination was higher for the most common haplotype, which produced more spores at 20 and 25°C. The isolate showed hyphal melanization at 5°C, which has been associated with survival and may be important because no resting structures have been described for F. circinatum. Our study determined that longer moisture periods during infection result in more severe disease from 7 to 24 h, regardless of the isolate virulence. This is the first study on virulence of the most abundant haplotypes of F. circinatum in Spain as affected by temperatures and moisture.

This study investigated the complex phenotypic and genetic response of Monterey pine (Pinus radiata) seedlings to co-infections by F. circinatum, the causal agent of pine pitch canker disease, and the oomycetes Phytophthora xcambivora and P. parvispora. Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations F. circinatum/P. xcambivora and F. circinatum/P. parvispora. Initially, seedlings inoculated only with F. circinatum showed less severe symptoms than seedlings co-inoculated or inoculated only with P. xcambivora or P. parvispora. However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with F. circinatum, showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, F. circinatum stimulated the up-regulation of all genes, while between the two oomycetes, only P. xcambivora induced significant up-regulations. In seedlings co-inoculated with F. circinatum and P.xcambivora or P. parvispora none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination F. circinatum/P.xcambivora and PDC in the combination F. circinatum/P. parvispora, thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism.

[EN] Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are pr

[EN] Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are pr

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.

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.

Hot water treatments (HWTs) were evaluated for their ability to reduce the incidence of Fusarium circinatum on Pinus radiata seeds. The effect of HWTs on conidial germination and mycelial growth of F. circinatum isolates representative of both mating types (MAT-1 and MAT-2) was determined by incubating conidial suspensions and agar plugs with mycelium in a hot water bath. The percentages of conidial germination and mycelial growth relative to the non-treated control decreased with increasing temperature and time of HWTs. MAT-2 isolates were more sensitive to HWTs than those of MAT-1, and no germination or mycelial growth of MAT-2 isolates was observed for temperatures above 50°C. The effect of HWT on P. radiata seed germination was also evaluated. The decrease in the percentage of seed germination compared with the non-treated seeds was always below 30 per cent for treatment temperatures <53°C. In addition, two naturally infected seed lots of P. radiata were used to assess the effect of HWTs on naturally infected seeds and on 3-month-old seedlings obtained from treated seeds. There was no infection detected in most of the treatments evaluated for either seed lot. Only 1 per cent of infection was detected on seeds treated with 50°C for 30 min in seed lot 1 and on seeds treated with 50°C and 52°C for 45 min in seed lot 2. The results obtained in this study show that HWTs at 51-52°C for 30 min can be used to reduce F. circinatum contamination on P. radiata seed. © 2012 Institute of Chartered Foresters. All rights reserved.

Hot water treatments (HWTs) were evaluated for their ability to reduce the incidence of Fusarium circinatum on Pinus radiata seeds. The effect of HWTs on conidial germination and mycelial growth of F. circinatum isolates representative of both mating types (MAT-1 and MAT-2) was determined by incubating conidial suspensions and agar plugs with mycelium in a hot water bath. The percentages of conidial germination and mycelial growth relative to the non-treated control decreased with increasing temperature and time of HWTs. MAT-2 isolates were more sensitive to HWTs than those of MAT-1, and no germination or mycelial growth of MAT-2 isolates was observed for temperatures above 50°C. The effect of HWT on P. radiata seed germination was also evaluated. The decrease in the percentage of seed germination compared with the non-treated seeds was always below 30 per cent for treatment temperatures <53°C. In addition, two naturally infected seed lots of P. radiata were used to assess the effect of HWTs on naturally infected seeds and on 3-month-old seedlings obtained from treated seeds. There was no infection detected in most of the treatments evaluated for either seed lot. Only 1 per cent of infection was detected on seeds treated with 50°C for 30 min in seed lot 1 and on seeds treated with 50°C and 52°C for 45 min in seed lot 2. The results obtained in this study show that HWTs at 51-52°C for 30 min can be used to reduce F. circinatum contamination on P. radiata seed. © 2012 Institute of Chartered Foresters. All rights reserved.