Testing systemic fungicides for control of Phytophthora oak root disease

Potassium phosphite (PP) formulations registered as fertilizers are now prohibited in Spain. Therefore, we evaluated the systemic fungicide fosetyl‐aluminium (fos‐al) in comparison with PP, against root rot caused by Phytophthora cinnamomi in Quercus woodlands. The direct effect of both systemic fungicides was evaluated in vitro on P. cinnamomi mycelial growth. Protection of cork and holm oak against infection was also evaluated in planta. Metalaxyl was included in both in vitro and in planta experiments for comparison purposes. At 100 μg/mL, PP totally inhibited colony radial growth, in comparison with 75% achieved by fos‐al. At doses recommended by manufacturers, with fos‐al and metalaxyl applications, root symptoms remained similar to the uninfected control levels. Based on these results, fos‐al is a candidate substitute product for PP in Quercus woodlands for control of Phytophthora oak root disease.     

Fungicide sensitivity of Dothistroma septosporum isolates in the UK

Dothistroma septosporum, a causal agent of Dothistroma needle blight (DNB), is a damaging fungal pathogen of pines that has recently started to affect native Scots pine woodlands in the UK. In addition to silvicultural methods, fungicide spraying of forest nursery stock can help prevent the spread of DNB. However, the effectiveness of modern single‐site fungicides against D. septosporum and the risk of fungicide resistance evolution remain largely unknown. In this project, we aimed to establish sensitivity profiles of D. septosporum to some widely used single‐site fungicide classes in vitro, and to determine whether fungicide resistance is already present, as this could increase the spread of D. septosporum genotypes on planting stock in native woodlands. For this purpose, we compared isolates of D. septosporum, originating from pine stands unexposed to fungicides, with isolates from nursery outbreaks, for sensitivity to a range of commonly applied fungicides. Most of the fungicides we tested were effective in vitro and we observed no significant shifts in sensitivity in forest nurseries. Although further tests in planta are required to confirm effectiveness of single‐site fungicides against D. septosporum, our results suggest that they can be successfully used in DNB control, although appropriate measures to prevent the evolution of fungicide resistance are strongly recommended.     

Efficacy of fungicides on Hymenoscyphus fraxineus and their potential for control of ash dieback in forest nurseries

Twenty‐six commercial formulations of fungicides at six concentrations were evaluated in vitro for their efficacy on mycelial growth of Hymenoscyphus fraxineus (anamorph Chalara fraxinea). The results are presented as EC₅₀, EC₉₀ and minimal inhibitory concentration values; the comparisons with the recommended application concentrations showed that 10 of the 26 fungicides were highly effective in their ability to inhibit the mycelial growth of the pathogen. The eight most effective fungicides identified based on multiple comparisons analysis were azoxystrobin, bitertanol, captan (in two‐component preparation with trifloxystrobin), difenoconazole, kresoxim‐methyl, spiroxamine (in multicomponent preparation with tebuconazole and triadimenol), tebuconazole and trifloxystrobin. Azoxystrobin, difenoconazole, kresoxim‐methyl, mancozeb, myclobutanil, pyrimethanil, tebuconazole and trifloxystrobin were selected to verify their effectiveness in in planta tests. During two field tests in nurseries, it was found out that tebuconazole (triazole), trifloxystrobin and kresoxim‐methyl (strobilurins) and mancozeb (dithiocarbamate) were significantly effective against H. fraxineus. The best results were gained with tebuconazole, when mean percentage of diseased saplings was 16.3%, whereas in the control, it was 63.6%. The combination and alternation of fungicides from the triazole, strobilurine and dithiocarbamate chemical groups should be an effective tool for protecting ash saplings in forest nurseries. Moreover, exact timing of the treatment reflecting rainfall and development of first symptoms in the foliage will be also important.     

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.     

Action in vitro de fongicides benzimidazoles et thiophanates sur Ceratocystis fimbriata E. et H. f. platani

In vitro action of selected benzimidazole and thiophanate systemic fungicides on Ceratocystis fimbriata E. et H. f. platani. The relative effects of several systemic fungicides (benzimidazoles: carbendazim, benomyl, and thiabendazole; and a thiophanate: methyl-thiophanate) of French strain of Ceratocystis fimbriata E. et H. (platani were studied in vitro by measuring mycelium diameter growth, percent conidiospore germination, and development from chlamydospores. Both strains were sensitive to carbendazim and methyl-thiophanate. These chemicals inhibited mycelium growth and development of chlamydospores.     

In vitro effects of four ectomycorrhizal fungi, Boletus edulis, Rhizopogon roseolus, Laccaria laccata and Lactarius deliciosus on Fusarium damping off in Pinus nigra seedlings

Species of Fusarium cause damping off of forest seedlings resulting in considerable losses and oftentimes fungicides are used to control the disease. Such chemicals may be detrimental to the environment. Protection of forest seedlings against this disease must be focused on integrated management for in which biological control is one of the most important tools. Mycorrhizal fungi may provide protection against pathogen invasion of seedlings. In this study, the interactions between the mycorrhizal fungi Boletus edulis, Rhizopogon roseolus, Laccaria laccata and Lactarius deliciosus and damping off pathogens (Fusarium oxysporum and F. moniliforme) were investigated in vitro and mechanisms involved in the protection of damping off in P. nigra seedlings are also discussed. The effect of filtrates from mycorrhizal species on spore germination of F. oxysporum varied throughout the tests. At the end of the assay, the inhibitory effect only could be observed in the L.␣deliciosus treatment. None of the filtrates reduced spore germination in F. moniliforme. Finally, three of the four mycorrhizal species grown for 2 months in the substrate, L. laccata, L.␣deliciosus and B. edulis, increased the survival of Fusarium inoculated P.␣nigra seedlings.     

Effect of fungicides,endophytes and fungal filtrates on in vitro growth of Spanish isolates of Gremmeniella abietina

The effect of eight fungicides and 15 endophytes isolated from twigs of healthy Pinus halepensis trees on the growth rate of four Spanish isolates of the pathogen Gremmeniella abietina was evaluated in vitro. In the fungicide experiments, four doses of each fungicide tested were added to the growth medium. In the endophyte experiments, dual cultures endophyte‐pathogen were paired in Petri dishes. Furthermore, growth of three G. abietina isolates was evaluated on malt agar with pine needle extract amended with filtrates from cultures of endophyte E14, which produced a brownish compound apparently inhibiting G. abietina growth. The results obtained suggested that chlorothalonil and daconil were the most suitable fungicides at low doses to reduce growth of G. abietina isolates from Spain. Four of the endophytes tested in vitro showed strong antagonistic activity against G. abietina and deserve further testing in vivo. The endophyte E14 produced in vitro a brownish compound which almost completely inhibited mycelial growth of G. abietina isolates from Spain.     

Visualizing the early infection of Agathis australis by Phytophthora agathidicida,using microscopy and fluorescent in situ hybridization

Phytophthora agathidicida (PTA) causes a root rot and collar rot of New Zealand kauri (Agathis australis). This study developed techniques to visualize early infection of kauri by PTA in deliberately inoculated seedlings. Conventional light microscopy was carried out on cleared and stained roots using trypan blue to observe PTA structures. Additionally, scanning electron microscopy (SEM) was used to study the PTA root structures at a higher resolution. A fluorescent in situ hybridization assay (FISH) was developed using a PTA‐specific probe to label PTA structures in planta. Infection progression in roots of 2‐year‐old kauri inoculated with PTA at 5, 10, 16 and 20 days post‐inoculation (d.p.i.) was compared using these three approaches. Light microscopy identified no Phytophthora‐like structures in the control treatments. In PTA‐inoculated plants, lignitubers were produced 5 d.p.i. in cortical cells. Infection was localized after 5 days, but as the infection progressed (up to 20 d.p.i.), the ‘degree’ of root infection increased, as did the number of replicates in which structures were observed. SEM provided higher resolution images; again, no PTA structures were observed in the negative control material examined. The slide‐based FISH‐specificity assay successfully hybridized with PTA hyphae. Fluorescence was observed using 330–380 nm excitation and an emission filter at 420 nm (DAPI), with PTA nuclei fluorescing a bright greenish‐yellow. Cross‐reactivity was not observed when the assay was applied to six other non‐target Phytophthora species. Successful hybridization reactions occurred between the primer and PTA structures in planta. Applying this FISH assay has allowed clear differentiation of the intracellular and intercellular structures of PTA. The technique can be applied to longer term studies or analysis of ex situ inoculation studies aiming to elucidate differential host‐responses to the pathogen. Additionally, the technique could be applied to study the interactions with other fungal endophytes (e.g. mycorrhizal fungi), which could be assessed for biocontrol potential as part of the integrated management of the disease.     

Testing the effect of routine fungicide application on ectomycorrhiza formation on Pinus halepensis seedlings in a nursery

The fungicides benomyl, captan, hymexazol, iprodione, propamocarb hydrochloride and thiram were applied in a combined schedule to Pinus halepensis seedlings grown under nursery conditions to study their effect on mycorrhiza formation and plant growth. The inoculated fungi Pisolithus tinctorius (strains 3SR and Mx) and Lactarius deliciosus formed mycorrhizas when fungicides were applied. However, fungicide application led to a significant decrease of mycorrhizal plants with Pisolithus tinctorius strain 3SR (from 62% without fungicides to 35% with fungicides). This effect could be attributed to a direct effect of fungicides on mycelial growth. The morphometric and nutritional parameters were very similar in plants treated with or without fungicides, and no differences on sanitary status of plants were noted. The use of preventive fungicide treatments in plant production, which may have a harmful effect on the environment, is therefore questioned.     

In vitro evaluation of fungicides against Cylindrocladium spp. causing diseases of Eucalyptus in Kerala,India1

A total of 22 fungicides were evaluated in vitro for their efficacy against Cylindrocladium quinqueseptatum (CQ), C. ilicicola (CI), C. floridanum (CF), C. parvum (CP), and C. camelliae (CC) causing various diseases of Eucalyptus in Kerala, India. Though there were a number of fungicides effective (ED₁₀₀) in the comdial germination and poisoned food techniques, only carbendazim provided complete inhibition of CQ, CI and CC in the soil-fungicide screening technique; carbendazim was also highly effective against CF and CP. C. ilicicola appeared to be more tolerant than other species of Cylindrocladium as only a few fungicides were found to be effective against it even in the conidial germination technique. On comparison of the three fungicidal evaluation techniques, it is concluded that, for a pathogen producing microsclerotia, like Cylindrocladium, the soil-fungicide screening technique is the most appropriate one for obtaining reliable results.     

Charcoal root and stem rot of Eucalyptus

Macrophomina phaseolina (Tassi) Goid. caused a serious root and stem rot in juvenile seedlings of Eucalyptus in nurseries. This is a new host record. The pathogen produced systemic infection in the seedlings. The symptoms and histopathology and factors influencing the disease were studied. Of five fungicides tried in the laboratory and in the field, bavistin and dithane M-45 at 0.2% showed promising results for the control of the disease.     

Fungicidal Control of Lophodermium seditiosum on Pinus sylvestris Seedlings in Swedish Forest Nurseries

During the 1990s, there were serious outbreaks of the pathogen Lophodermium seditiosum on pine seedlings in Swedish forest nurseries, even though the seedlings had been treated with the fungicide propiconazole. The present experiment was carried out to evaluate two other fungicides, fluazinam and azoxystrobin, as possible alternatives to propiconazole. In the tests, which were all carried out in the same forest nursery, seedlings were treated with either propiconazole, fluazinam or azoxystrobin, and the proportion of needles with ascocarps of L. seditiosum and the number of ascocarps per needle were recorded over the following 2 yrs. Seedlings treated with azoxystrobin already appeared healthier than control seedlings in September of the first year, and by November all azoxystrobin-treated seedlings had fewer ascocarps per needle compared with control seedlings. In autumn of the second year, there were no ascocarps on seedlings treated with fluazinam or azoxystrobin, whereas seedlings treated with propiconazole had similar numbers of ascocarps to non-treated control seedlings.     

Effects of oak root pruning in forest nurseries on potential pathogen infections

Despite the general practice of root pruning, little is known about the potential impact of reducing shoot/root systems of oak seedlings in this way on their future susceptibility to pathogens, for example Cylindrocarpon spp., Fusarium spp., Ilyonectria spp., Pythium spp. Phytophthora spp. or Rhizoctonia spp. In this study, root‐pruned and non‐pruned seedlings of Quercus robur grown under controlled conditions were inoculated with aggressive and less‐aggressive pathogens. Results indicated, in contrast to our initial assumption, that pathogens significantly reduced lateral root biomass more in non‐pruned seedlings, the extent of the response depending on the pathogens species. In response to pathogen pressure, pruned seedlings tended to attain a higher dry stem mass fraction, but lower dry leaf mass fraction. Pathogens also suppressed leaf mass in total root dry mass fraction (dry leaf mass/total root dry mass ratio, in g × g^?1) more in pruned than in non‐pruned seedlings. These results suggest differences in growth between non‐pruned and pruned seedlings in response to pathogen stress. In nurseries, root pruning of oak trees may enhance the reduction in leaf mass in lateral roots mass fraction resulting from pathogen infections, which may decrease seedling quality. It is therefore important to ensure a low level of inoculum of soil‐borne pathogens to minimize the risk of seedling infection.     

Enhancing resistance in Pinus radiata seedlings to terminal crook (Colletotrichum acutatum) using methyl jasmonate and ultraviolet‐C radiation

Colletotrichum acutatum is a fungal pathogen that causes terminal crook disease in radiata pine (Pinus radiata) seedlings in New Zealand forest nurseries. Symptoms of infection include malformation or death of the growing tip and a stiffening and thickening of the stem. Although the disease can be managed effectively using fungicides, the New Zealand forest industry is interested in alternative control options such as induced resistance. The aim of this study was to evaluate spray application of chitosan (1.4 g/l) or 2.25 mm methyl jasmonate (MeJA) and irradiation with UV‐C (2.16 kJ/m²) for their potential to induce resistance to terminal crook. The treatments were applied to 4‐month‐old seedlings at 1 week before pathogen inoculation. By the end of the experimental period (42 days after inoculation), there was 80% disease incidence in the controls, with 48% of seedlings exhibiting severe terminal crook symptoms. The most effective treatment (p < 0.05) was MeJA with 16% disease incidence and none with severe symptoms. UV‐C also significantly (p < 0.05) reduced infection with 52% incidence and 20% of seedlings exhibiting severe symptoms. Chitosan did not reduce disease incidence (72%) compared with the control (80%) but did significantly reduce (p < 0.05) disease severity with 28% exhibiting severe symptoms. MeJA was the only treatment that significantly (p < 0.05) reduced the detrimental effects of infection on seedling apical growth and stem diameter. To our knowledge, this is the first report demonstrating the potential for MeJA and UV‐C to control terminal crook in radiata pine.     

Surveys for asymptomatic persistence of Sphaeropsis sapinea on or in stems of red pine seedlings from seven Great Lakes region nurseries

The fungus Sphaeropsis sapinea persists on or in stems of asymptomatic red pine (Pinus resinosa) nursery seedlings, and proliferates to cause collar rot and mortality after planting. In the spring of 2002, seven nurseries were surveyed to determine the potential range in frequency of asymptomatic persistence: three operated by Wisconsin Department of Natural Resources (DNR), two by Minnesota DNR, one by Michigan DNR, and one by USDA Forest Service (in Michigan). At each nursery five groups of 20 asymptomatic red pine seedlings were collected near an inoculum source (red pine windbreak), if present, and five groups of 20 asymptomatic seedlings were collected away from such a source (1400 seedlings total). A segment of the lower stem/root collar from each seedling was surface disinfested and incubated on tannic acid agar. Transfers were made from resulting colonies and the pathogen identified from pycnidia and conidia produced in culture. The pathogen was identified from asymptomatic seedlings collected in all Wisconsin and Minnesota nurseries, but was never detected from seedlings from the Michigan DNR or USDA Forest Service nurseries. Frequencies of detection were greater (as high as 88%) from asymptomatic seedlings near red pine windbreaks including diseased trees than from seedlings distant from such windbreaks. A subset of isolates from asymptomatic seedlings was characterized using inter‐simple sequence repeat–polymerase chain reaction analysis. Most isolates were the A group of S. sapinea, but B group isolates (recently named Diplodia scrobiculata) were also obtained from one nursery. One Minnesota nursery was more extensively sampled in 2003, with 17–44 groups of five asymptomatic red pine seedlings collected in four separate fields (525 seedlings total). The mean frequency of detection of the pathogen in these four fields ranged from 40 to 71%. Persistence of S. sapinea on or in asymptomatic seedlings continues to be problematic, not only because of the potential for subsequent seedling mortality, but also as a means by which a pathogen may be widely distributed.     

Pathogenicity of seed‐associated fungi to Podocarpus falcatus in vitro

Twenty‐nine fungi that were isolated from seeds and female cones of Podocarpus falcatus from natural forests in Ethiopia, were assessed for their impact on seeds and seedlings of the same host. Based on the results from in vitro seed inoculation tests, we could group the fungi into five categories as: (i) isolates that were pathogenic only to seeds and had no obvious impacts on the germlings; (ii) isolates that were pathogenic only to the germlings; (iii) isolates that were pathogenic both to seeds and the emerging germlings; (iv) isolates that were more or less harmless; and (v) isolates that were germination promoters. Inoculation tests were also performed on 4–7‐day‐old aseptically grown seedlings. Fusarium oxysporum and Polyporus sp., were strongly pathogenic to both seeds and seedlings, while Nectria gliocladioides, Peniophora cinerea and Pestalotiopsis neglecta also demonstrated pathogenicity but to a lesser extent. Other isolates, e.g. Diaporthe spp. resulted in increased germination of P. falcatus seeds and no pathogenicity to seedlings. However, further investigations are required in order to find out how these fungi behave under nursery or field conditions.     

Risk from Sirococcus conigenus to understory red pine seedlings in northern Wisconsin

Interest in development of multicohort stands of red pine (Pinus resinosa) in the Great Lakes region of North America prompted an investigation of the potential impact of the shoot blight pathogen Sirococcus conigenus (syn. S. strobilinus) on understory red pine seedlings. In May 2002 and 2003 healthy, 1‐year‐old red pine seedlings were planted in the understory of a maturing red pine plantation in northern Wisconsin in an area with a history of presence of this pathogen. Occurrence of shoot blight symptoms was recorded periodically during the summer, and in each year seedlings were harvested in fall and examined for signs of shoot blight pathogens. By fall 2002 and 2003, respectively, shoot blight incidence was 89% and 98% and most seedlings were dying. Pycnidia with conidia of S. conigenus were present on almost all of the symptomatic seedlings. The conifer shoot blight and canker pathogen Diplodia pinea (syn. Sphaeropsis sapinea) was also detected, though less frequently. Pycnidia of S. conigenus tended to be found more frequently on symptomatic current year's shoots than symptomatic previous year's shoots; the opposite was true for pycnidia of D. pinea. Risk from S. conigenus to understory red pine seedlings should be considered in any plans for development of multicohort red pine stands in areas where the pathogen is present.     

Effects of associated fungi Sclerophoma pythiophila and Cenangium ferruginosum on Gremmeniella abietina dieback in Spain

The interactions between Gremmeniella abietina and either Sclerophoma pythiophila or Cenangium ferruginosum, fungi frequently isolated from diseased twigs along with G. abietina, were studied under laboratory (dual cultures) and greenhouse conditions (double‐inoculations). Virulence of each species was also evaluated in greenhouse experiments by means of single‐inoculations. In vitro interactions were assessed on Petri dishes containing malt agar with pine needle extract, and greenhouse experiments were performed on 1‐year‐old Pinus halepensis seedlings. In vitro growth of G. abietina was inhibited by both fungi when grown in dual culture. In single‐inoculations, G. abietina caused the greatest necrosis length on P. halepensis seedlings, followed by S. pythiophila, whereas C. ferruginosum did not cause significant necrosis. In double‐inoculations, C. ferruginosum was able to reduce the length of necrosis caused by G. abietina on the P. halepensis seedlings. In contrast, necrosis length was greater in seedlings inoculated with both S. pythiophila and G. abietina than in those inoculated with G. abietina alone. Therefore, S. pythiophila seems to play a role in disease expression caused by G. abietina on P. halepensis in Spain.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.