Conifer seedling root fungi and root dieback in Finnish nurseries

Fungi were isolated from the roots and growth substrate of bare‐rooted and containerized Pinus sylvestris and Picea abies nursery seedlings displaying a root dieback. Isolations were also made from visually healthy seedlings. The potential pathogenicity of all isolated species was determined in laboratory trials.

Cylindrocarpon spp., Fusarium spp. and Trichoderma viride were frequently isolated. The isolation frequency of a uninucleate Rhizoctonia‐like fungus, Pythium spp. and Phytophthora imdulatum from diseased containerized seedlings and their pathogenicity in tests suggest that these fungi are likely involved in the root dieback disease in containers. The pathogenic Rhizoctonia‐like fungus in addition to Pythium spp. was also isolated from bare‐rooted seedlings. In greenhouse tests Pythium spp. were more pathogenic to 4‐week‐old Scots pine seedlings grown before transplantation in unsterile substrate than to those seedlings grown axenically in agar. External factors are considered to have some role in the expression of disease.     

Incidence of dieback disease following fungal inoculations of sexually and asexually propagated shisham (Dalbergia sissoo)

Shisham (Dalbergia sissoo) is an important multipurpose tree with great economic importance, but this tree has been devastated by dieback disease. Seedlings and asexually propagated (cuttings) plants were artificially inoculated with four fungi (Fusarium solani, Botryodiplodia theobromae, Curvularia lunata and Ganoderma lucidum) to evaluate the potential role of these fungi in shisham dieback disease. Results at 2 years revealed that highest disease was caused by inoculation of F. solani (31.39%), followed by B. theobromae (19.042%) and C. lunata (12.22%), but no dieback disease was caused by G. lucidum. During both years, seedlings exhibited greater susceptibility to disease (17.24%) compared to cuttings (7.83%). In particular, F. solani caused more disease in seedlings (46.18%) compared to cuttings (16.61%). With the F. solani inoculations, maximum disease rate was observed at 8 weeks post‐inoculation both in seedlings (77%) and in cuttings (31%), but the maximum disease increase was observed at 4–5 weeks post‐inoculation. Analysis of variance showed significant differences among the different fungi and also between seedlings and cuttings. F. solani can be considered as a major fungal pathogen contributing to dieback disease of shisham, and asexual propagation can reduce the severity of dieback.     

Infection of Norway spruce container seedlings by Gremmeniella abietina

First‐ and second‐year containerized Norway spruce seedlings were inoculated with conidia of type A (large tree type) and type B (small tree type) of Gremmeniella abietina var. abietina at different times during the summer. The appearance of symptoms after artificial inoculation and natural infection on spruce seedlings were recorded the following spring and compared with the disease symptoms on Scots pine seedlings. The proportion of diseased seedlings after inoculation reached as high as 80%. The susceptible period during the summer began later on the first‐year seedlings than on the second‐year seedlings, and was similar for the pine seedlings. Susceptibility of first‐year seedlings was highest in August and on second‐year seedlings in July. The accumulated temperature sum, relative humidity and height growth for first‐ and second‐year seedlings was assessed. Natural infection in 2002 caused more disease on pine than on spruce seedlings. Experimental thinning of seedlings had no effect on disease incidence. In a preliminary comparison between the ability of A and B types to cause disease in Norway spruce seedlings, type B caused more damage than type A after inoculation. However, type A caused a high disease frequency in other experiments in this study. Symptoms on Norway spruce seedlings often first occurred in the mid‐section of the shoot, and were similar to those observed on pine seedlings: needles turned brown, starting at the needle base, in the spring following inoculation. On first‐year spruce, diseased needles were shed rapidly, in contrast to a slower rate of shedding on first‐year pine seedlings. Pycnidia developed about 2 years after inoculation (on pine 1 year after inoculation). On Norway spruce seedlings the lower part of the shoot, including the lateral shoots, often remained alive. The experiments show that G. abietina can cause disease on containerized Norway spruce seedlings under nursery conditions in Finland. The coincidence of spore dispersal, seedling susceptibility and predisposing factors are important in disease development.     

Higher resistance of the offspring of Scots pine trees resulting from natural regeneration in old foci of Heterobasidion annosum root rot

Abstract

Heterobasidion annosum (Fr.) Bref. is a fungal pathogen causing annosum root rot – one of the most economically important diseases in coniferous stands. The major aim of this study was to compare the resistance of the offspring of Scots pine trees (Pinus sylvestris L.) from seed orchards and the offspring of trees that were naturally reproduced in old foci of the disease. In experiments conducted in vitro, we used 960 seedlings which were grown from seeds collected from 60 trees in 6 old foci of the disease and 640 seedlings grown from seeds collected in 4 seed orchards. The offspring of trees from seed orchards after inoculation with H. annosum had nearly twofold higher mortality rate than the seedlings developed from the seeds collected in old foci of the disease. This suggests that the offspring of self-sown trees in old foci of the disease has a greater, genetically conditioned resistance to annosum root rot.     

First report of Fusarium solani causing wilt of Melia dubia

Melia dubia, a multipurpose tree species, is gaining importance to meet the demand supply gap of timber, plywood and pulpwood . In June 2016, a serious outbreak of wilt disease was observed in M. dubia seedlings planted in the Central Nursery of Forest Research Institute (FRI), Dehradun, India. The disease led to the destruction of one hundred thousand (100,000) seedlings. Earlier in June 2012, serious wilting of M. dubia seedlings was observed in Haryana, India. The pathogen was identified as Fusarium solani following standard laboratory procedures and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). The pathogenicity of three isolates has been proved under greenhouse conditions. This is the first report of F. solani causing wilt of M. dubia.     

Effects of elevated atmospheric CO<Subscript>2</Subscript> on two southern forest diseases

Research into the effects of rising atmospheric carbon dioxide (CO₂) on plant diseases remains limited despite the economic importance of this subject. Loblolly pine (Pinus taeda) seedlings were exposed to ambient and twice ambient levels of atmospheric CO₂ prior to inoculation with the fusiform rust fungus (the obligate pathogen Cronartium quercuum f.sp. fusiforme, CQF) or the pitch canker fungus (the facultative pathogen Fusarium circinatum, FC). Additionally, northern red oak seedlings (Quercus rubra; an alternate host of CQF) were exposed to ambient or elevated levels of atmospheric CO₂ prior to inoculation with CQF. In all cases, disease incidence (percent of plants infected) and disease severity (proportion of each plant affected) were determined; with the oak seedlings, the latent period (time to sporulation) was also monitored. In general, disease incidence was decreased by exposure to elevated CO₂. This exposure also increased the latent period for CQF on oak seedlings. In no instance did exposure to elevated CO₂ affect disease severity. This research demonstrated that plants may benefit from exposure to the increasing concentration of CO₂ in the atmosphere through decreases in fungal disease incidence.     

Effect of uninucleate Rhizoctonia on Scots pine and Norway spruce seedlings

One‐year‐old container‐grown seedlings were planted in spring on clear cut areas: the Norway spruce (Picea abies) on a moist upland site (Myrtillus‐type) and Scots pine (Pinus sylvestris) on a dryish upland site (Vaccinium‐type). While still in the nursery, half of the seedlings of each species had been inoculated during the previous summer, with a uninucleate Rhizoctonia sp., a root dieback fungus. At outplanting all the seedlings appeared healthy and had a normal apical bud, although the height of the inoculated seedlings was less than that of the uninoculated control seedlings. At the end of the first growing season after planting, the mortality of inoculated Scots pine and Norway spruce seedlings was 25 and 69%, respectively. After two growing seasons the mortality of inoculated seedlings had increased to 38% for Scots pine and 93% for Norway spruce. The mortality of control seedlings after two growing seasons in the forest was 2% for Scots pine and 13% for Norway spruce. After outplanting the annual growth of inoculated seedlings was poor compared with the growth of control seedlings. These results show that, although Rhizoctonia‐affected seedlings are alive and green in the nursery, the disease subsequently affects both their survival and growth in the forest.     

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.     

Inhibition of the regeneration of Japanese black pine (<Emphasis Type="Italic">Pinus thunbergii</Emphasis>) by black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) in coastal sand dunes

Pine forests are declining because of pine wilt disease and Robinia pseudoacacia, a nitrogen-fixing species, is dominating coastal forests along the Sea of Japan. We examined the effects of R. pseudoacacia on the regeneration of a native pine species, Pinus thunbergii. Two 200 m² plots were set up at the border of a P. thunbergii and R. pseudoacacia-dominated area in a coastal forest. We conducted monthly censuses of emergence, distribution and survival of pine seedlings in the plots from May 2003 to December 2004. Light intensity and soil properties were also measured to analyze the relationships between the survival of pine seedlings and environmental conditions using the Mantel test and the structural equation model. Pinus thunbergii seedlings emerged in spring–early summer and in late autumn. Survival of pine seedlings in the R. pseudoacacia-dominated subplots was less than half that in the pine-dominated subplots. Survival of pine seedlings emerging in May 2003 was significantly reduced by the lower light intensity and higher soil nitrogen in R. pseudoacacia-dominated subplots. The tendency was the same for seedlings emerging from April to May 2004. We concluded that R. pseudoacacia reduced the intensity of light during the growing season and increased the nitrogen content of soil, which resulted in inhibition of the natural regeneration of P. thunbergii.     

Infection experiments with Gremmeniella abietina on seedlings of Norway spruce and Scots pine

Conidia of Gremmeniella abietina infected and caused disease symptoms in annual shoots of both Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings. In Norway spruce shoots the infection remained largely latent, with only a few seedlings showing symptoms. Mycelial growth inside the shoots was faster in Scots pine than in Norway spruce and was favoured by low temperature in both hosts. The shoots of Norway spruce seedlings had higher endophyte populations than those of Scots pine, and the populations were decreased by low temperatures. Reductions in the normal epiphytic or endophytic flora by acid mist treatments seemed to favour the development of G. abietina.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Infection of Scots pine seedlings by Gremmeniella abietina during summer under different inoculum potential

The connection between natural conidia dispersal of Gremmeniella abietina (A‐type) and the disease incidence and severity in first and second year conifer seedlings of Pinus sylvestris was studied in central Finland. The seedlings where exposed to natural infection for a 3‐week period throughout the growth season, followed by a 3‐week incubation period in a growth chamber to promote symptom expression. In second year seedlings the infection periods in May–June had a similar effect as regards disease outbreak and roughly half of the successful infections occurred during these periods. However, the first year seedlings were more severely diseased after the infection period in late July/early August, although the inoculum density was lower during this time compared with the period in late June/early July. This result is consistent with earlier studies. The effect of age and growth phase of the seedlings, temperature sum and the number of conidia on the disease occurrence is discussed and compared with the results of earlier studies in which artificial inoculation was employed.     

Artificial inoculation and susceptibility of Pinus armandii to Dothistroma septosporum

Dothistroma needle blight (DNB) is a serious needle disease of conifers that primarily affects pine species (Pinus spp.). Dothistroma septosporum is one of the DNB pathogens that has a diverse range of host species excluding Pinus armandii. In 15 inoculated P. armandii seedlings, D. septosporum acervuli were observed in 43 infected needles of ten seedlings with a mean disease severity of 1.11% at 25 weeks after inoculations, demonstrating the potential of D. septosporum to cause symptoms on the needles of P. armandii via artificial inoculation. The disease severity of P. armandii was similar to the positive control, Pinus nigra (median 0.75 for P. armandii to 0.70 for P. nigra), thus, P. armandii acts under artificial conditions as a susceptible host species.     

Virulence of Fusarium oxysporum and F. commune to Douglas‐fir (Pseudotsuga menziesii) seedlings

Fusarium species can cause damping‐off and root rot of young conifer seedlings, resulting in severe crop and economic losses in forest nurseries. Disease control within tree nurseries is difficult because of the inability to characterize and quantify Fusarium spp. populations with regard to disease potential because of high variability in isolate virulence. Fusarium isolates were collected from healthy and diseased seedlings of Douglas‐fir (Pseudotsuga menziesii) and western white pine (Pinus monticola) from a nursery in Idaho, USA. Molecular markers such as DNA sequences (mitochondrial small subunit and nuclear translation elongation factor 1‐alpha) and amplified fragment length polymorphism were used to identify isolates as either F. oxysporum or F. commune. In addition, diagnostic primers were developed to detect and distinguish F. commune from F. oxysporum. In vitro and greenhouse virulence tests were completed on Douglas‐fir germinants and seedlings. For Douglas‐fir germinants and seedlings, F. oxysporum isolates generally caused less severe symptoms, whereas most F. commune isolates caused mortality through damping‐off. This is the first report of direct evidence that F. commune can cause damping‐off disease on Douglas‐fir seedlings under greenhouse conditions.     

Effects of inoculum density of pinewood nematode on the development of pine wilt disease in Japanese black pine seedlings pretreated with simulated acid rain

Six‐month‐old Japanese black pine seedlings (Pinus thunbergii) were exposed to simulated acid rain (SAR) at pH 3 and 2 three times a week. After treatment for 2 months, the seedlings were inoculated with a virulent isolate (S10) of the pinewood nematode (Bursaphelenchusxylophilus), at three inoculum levels (P_i = 50, 160 or 500 nematodes per seedling). In seedlings inoculated with 500 nematodes, both population growth of nematodes and disease development were accelerated by pretreatment with SAR at pH 3 or 2. In seedlings inoculated with 50 nematodes, population growth of the nematodes was suppressed and more time was needed for seedlings to die when pretreated with pH 3 SAR. This suggests that exposure to pH 3 SAR increased not only the progress of mortality, but also simultaneously enhanced the tolerance limit of the seedlings to the pinewood nematode – the critical value of physiological burden (represented as a product of time and initial nematode population) necessary to kill a seedling. Exposure to pH 2 SAR accelerated nematode reproduction in seedlings and increased seedling mortality irrespective of the number of nematodes inoculated.     

Effect of inoculum density of Macrophomina phaseolina on seedling susceptibility of six conifer species

The severity of charcoal root disease of conifer seedlings (Abies concolor, A. magnified, Pinus jeffreyi, P. lambertiana, and Pseudotsuga menziesii) was a power function of the sclerotial density of the fungus (Macrophomina phaseolina) in nursery soil.     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

Identification of Cylindrocladium sp. causing damping-off disease of Japanese black pine (Pinus thunbergii) and factors affecting the disease severity in a black locust (Robinia pseudoacacia)-dominated area

The regeneration of Japanese black pine (Pinus thunbergii) seedlings is inhibited in a black locust (Robinia pseudoacacia)-dominated area. We examined the presence of pathogenic fungi in Japanese black pine seedlings in the area in order to determine the effect of pathogenic fungi on the inhibition of regeneration. When Japanese black pine seedlings were planted in the soil obtained from a black locust-dominated area, all of the seedlings died under low-intensity light conditions, whereas 84% of the seedlings survived in the soil obtained from a Japanese black pine-dominated area under the same light conditions. One fungus was isolated from 48.7% of the dead pine seedlings and was identified as Cylindrocladium pacificum Kang, Crous & Schoch, based on the morphological characteristics, growth, and DNA analysis. This fungus was also isolated from 50% of the dead pine seedlings in 2005 and 66.7% of the seedlings in 2006—both were planted in a black locust-dominated area. The virulence of this fungus increased under high-nitrogen and/or low-intensity light conditions. These results reveal the possibility that the soil eutrophication and shading by the black locust are conducive to a severe damping-off disease and threaten the survival and regeneration of Japanese black pine seedlings.     

Inhibiting factors of symptom development in several Japanese red pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) families selected as resistant to pine wilt

Pinus densiflora and P. thunbergii, native to Japan, are highly susceptible to pine wilt disease caused by infection with a pine wood nematode (Bursaphelenchus xylophilus). Trees of these susceptible species have occasionally been found surviving in forests that are extensively damaged by this disease. Seedlings from a part of surviving trees that were selected as resistant families indicate lower mortality rates after the infection. The factors that prevent the symptoms from developing in resistant families of a susceptible species, P. densiflora, as based on the analysis of the pathogens behavior in the tree tissue and the anatomy of the resistant families, are presented in this paper. Nematode populations remained lower in the stems of seedlings from resistant families of P. densiflora than in the stems of non-resistant families. Areas dysfunctional in water transport developed in the stems of resistant families, but did not reach a size large enough to seriously block the ascent of sap. These results suggest that there are systems within the seedlings that prevent nematode migration and reproduction. The 2-year-old seedlings from resistant families of P. densiflora, however, did not suppress the pathogen activity. Numerous branches are a visible characteristic in the seedlings of some resistant families. The arrangement of the resin canals, the only channels in the tree to the pathogen migration, was disoriented at the joints between the branches and the main stem. Such a structure may be effective as a barrier to nematode dispersal.     

Hymenoscyphus pseudoalbidus and Hymenoscyphus albidus: viridiol concentration and virulence do not correlate

Hymenoscyphus pseudoalbidus is the causal agent of ash dieback, a disease that is presently endangering Fraxinus spp. throughout most of Europe. The phytotoxin, viridiol, was previously isolated from culture extracts of H. pseudoalbidus and found to be toxic to leaves of F. excelsior. Thus, we were interested in learning to what extent viridiol is responsible for pathogenicity of H. pseudoalbidus and investigated this using twelve isolates of H. pseudoalbidus. We also included five isolates of the closely related avirulent species, Hymenoscyphus albidus, in our studies. Some, but not all, isolates of H. pseudoalbidus and H. albidus produced measurable quantities of viridiol in culture. Three tests were used to determine to what extent viridiol concentration correlates with virulence: culture extracts were tested for activity in leaf segment tests and for inhibition of germination of seedlings of Fraxinus excelsior; virulence of the isolates was tested following infection of axenically cultured ash seedlings. Activity of the culture extracts varied, as did virulence of the isolates following inoculation into seedlings. No correlations were found between viridiol concentration and activities of culture extracts in leaf segment tests or in the germination test, nor between viridiol concentration and disease symptoms when inoculated into seedlings. However, activities of culture extracts in leaf segment and in the germination test correlated, as did the results of each of these tests with virulence in the infection experiment. Apparently, as yet unidentified factors other than the concentration of viridiol play important roles in the virulence of H. pseudoalbidus.