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.     

The occurrence and pathogenicity of uni- and binucleate Rhizoctonia and Pythiaceae fungi among conifer seedlings in Finnish forest nurseries

Uni- and binucleate Rhizoctonia and Pythiaceae fungi were isolated from roots of dieback seedlings. Only binucleate Rhizoctonia strains were present in the roots of healthy seedlings. In pathogenicity tests, uninucleate Rhizoctonia sp. were pathogenic and binucleate Rhizoctonia spp. non-pathogenic. Pythium ultimum var. ultimum and Phytophthora undulata were the only Pythiaceae fungi that killed 12-week-old seedlings.     

Aggressiveness of Fusarium oxysporum and F. verticillioides isolates on stone and scots pine under greenhouse conditions

Scots pine (Pinus sylvestris) and Stone pine (Pinus pinea) are two important species used in re-forestation that are subject to damage by damping-off fungi in forest nurseries. Twenty-two isolates of Fusarium oxysporum and F. verticillioides from diseased seedlings of eight different hosts were tested for aggressiveness on seeds and seedlings of both pine species, including the effects on seedling emergence and mortality. Scots pine was more susceptible to damping-off than Stone pine, as indicated both by reduced seedling emergence and elevated seedling mortality. The impact of F. oxysporum and F. verticillioides on seeds and seedlings did not differ significantly for either pine species. Our findings support previous studies that found that these are damping-off pathogens on the studied pines. Whereas most isolates proved to be pathogenic, some isolates of both Fusarium species showed to be non-pathogenic.     

Phytopbthora cactorum on walnut seedlings in Italian nurseries

Phytophthora cactorum was consistently isolated from basal stem tissues of walnut seedlings affected by collar and root rot. The isolates were identified on the basis of morphological and cultural characteristics and of electrophoretic patterns in polyacrylamide slab gels of total native mycelial proteins and isozymes (arylesterase, malate dehydrogenase, acid and alkaline phosphatase). The electrophoretic phenotypes of walnut isolates were identical or very similar to those of P. cactorum isolates from different hosts (apple, loquat, pineapple-guava and strawberry). In pathogenicity tests, P. cactorum isolates from walnut proved to be highly pathogenic for seedlings of English walnut (Juglans regia) and moderately pathogenic for seedlings of eastern black walnut (Juglans nigra); moreover, they did not rot apple seedlings, suggesting host-specificity. Poor soil drainage and excessive irrigation can be considered as predisposing factors to collar and root rot incited by P. cactorum.     

Fungi associated with the fir bark beetle Cryphalus piceae in Poland

The aim of this study was to identify fungi associated with Cryphalus piceae on European silver fir (Abies alba) in Poland and to test the pathogenicity of selected isolates. Fungi were isolated from five populations of overwintered adults and their galleries. A great diversity of taxa was associated with C. piceae. In total, 2487 isolates, including 58 species distributed in 25 genera, were obtained. The two most frequently isolated fungi, an undescribed species of the genus Geosmithia and Ophiostoma piceae, appeared to be specifically associated with C. piceae, whereas Pesotum fragrans, Pesotum sp. and Sporothrix sp. were sporadically associated. Two‐year‐old seedlings of silver fir were wound‐inoculated with three species of fungi (Geosmithia sp., O. piceae and Pesotum sp.) recovered from C. piceae. Only Pesotum sp. showed pathogenic ability, but we do not consider it to be an important pathogen of A. alba.     

Studies on anastomosis groups of Rhizoctonia solani isolates causing disease in two forest nurseries in Poland

Thirty‐eight isolates of Rhizoctonia spp. were isolated from Scots pine (Pinus sylvestris) seedlings with damping‐off symptoms, originating from two forest nurseries in central‐west Poland (Wronczyn and Jarocin) and from diseased seedlings grown in soil from Wronczyn nursery. Majority of these isolates (79%) had multinucleate cells and were identified as Rhizoctonia solani. The remaining isolates were recognized as binucleate Rhizoctonia spp. R. solani isolates were characterized using hyphal anastomosis and were divided into five anastomosis groups (AG). The most prevalent was AG5 (37% of isolates), followed by AG2‐1 (30%) and 27% of the isolates were identified as AG4. Groups AG1‐IB and AG2‐2 were only represented by single isolates. The virulence recorded as mortality (in percentage) was comparatively high for binucleate and multinucleate isolates of Rhizoctonia spp. Sequence analysis of the polymerase chain reaction (PCR)‐amplified internal transcribed spacer (ITS) rDNA region was used for phylogenetic analysis. The dendrogram showed that isolates were distinctly separated based on their AG types and there was no relationship between pathogenicity on Scots pine seedlings and the AG to which the isolates belong to. The results are discussed with respect to pathogenic potential of the various AG groups.     

Fungi colonizing Scots-pine cone scales and seeds and their pathogenicity

Fungi were isolated from the cone scales and seeds of Scots pine using plating on malt-extract agar and/or the standard blotter method in a Jacobsen's apparatus. Alternaria alternata, Epicoccum purpurascens and Ulocladium atrum were isolated from damping-off seedlings germinating on agar or filter paper, but, in pathogenicity tests with peat-sand (1:3) growth substrate, they were not pathogenic. All the Fusarium species isolated were pathogenic in growth substrate. Some F. avenaceum colonies formed no aerial hyphae and they proved to be a mixed culture of the fungus, a fluorescent Pseudomonas sp., and a gram-negative bacterium. The bacterial associates appeared to increase the pathogenicity of F. avenaceum. All the micro-organisms tested were more pathogenic in sterilized than in unsterilized peat-sand substrate.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

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.     

Molecular characteristics and functional analysis of the β‐1,4‐endoglucanase Bm‐eng‐1 gene of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae)

The pine nematode Bursaphelenchus mucronatus has been associated with pine wilt in China. This work was intended to investigate the role of the pathogenicity‐related β‐1,4‐endoglucanase gene of B. mucronatus in the infection of pines. In this study, the full‐length cDNA of the Bm‐eng‐1 gene was cloned and characterized from a B. mucronatus isolate. Phylogenetic analysis suggested that the Bm‐eng‐1 gene of B. mucronatus may be acquired from fungi through horizontal gene transfer. The function of the gene was demonstrated using RNA interference. RNA interference indicated that Bm‐eng‐1 was involved in the dispersal, reproductive ability and pathogenicity of B. mucronatus. In situ hybridization showed that Bm‐eng‐1 was specifically expressed in the oesophageal gland of B. mucronatus. Furthermore, to investigate the relation between β‐1,4‐endoglucanase activities and virulence of different isolates, the β‐1,4‐endoglucanase activities and the expression levels of Bm‐eng‐1 were detected in six B. mucronatus isolates with different virulence. β‐1,4‐Endoglucanase activity was generally higher in pathogenic isolates than in non‐pathogenic isolates, and the Bm‐eng‐1 expression levels in different isolates were positively correlated with the β‐1,4‐endoglucanase activity. These findings suggested that β‐1,4‐endoglucanase plays important roles in the pathogenic process of B. mucronatus, and the differential expression of the gene may underlie the different activity levels of β‐1,4‐endoglucanase, subsequently influencing variations in virulence of B. mucronatus isolates.     

Effects of osmopriming and hydropriming on vigour and germination of China aster (Callistephus chinensis (L.) Nees.) seeds

The aim of this research was to evaluate the effects of osmopriming and different hydropriming treatments on the vigour and germination of China aster (Callistephus chinensis) seeds. Seed vigour and germination tests were conducted at 10, 20 and 30°C in darkness for untreated, osmoprimed and hydroprimed seeds. The following parameters and categories of seeds and seedlings were evaluated: the mean germination time (MGT), T10, T50, U75–25, U90–10, the percentage of germinating seeds (Gmax), germination capacity, percentage of abnormal seedlings and dead seeds. The results showed that osmopriming accelerated seed germination to the largest extent and improved the uniformity of germination at 10 and 30°C. Among the hydropriming treatments the highest speed of germination was observed for seeds hydrated in 500 μL of water per 1 g of seeds for 48 h at 15°C. This treatment accelerated seed germination at 10°C compared with the control. Osmopriming increased seed germination capacity at 30°C. None of the applied hydropriming treatments improved this parameter.     

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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.     

Pathogenicity of Spanish isolates of Heterobasidion annosum s. s. in Pinus pinaster seedlings

The aim of this study was to test the pathogenicity of two Spanish isolates of Heterobasidion annosum sensu stricto in 2‐year‐old Pinus pinaster seedlings. Two types of inocula (woodchips and sawdust) were used to infect the seedlings by two different routes (stem inoculation and soil infestation). The mortality rates of the stem‐inoculated seedlings differed significantly from controls, but those of the seedlings infected via soil infestation did not differ. For both types of inoculation, the lesions were longer, and wilting symptoms were more severe in the seedlings inoculated with H. annosum than in control seedlings. For stem inoculation, biomass allocation did not differ significantly between the infected and control seedlings. However, the percentage of fine roots was lower in seedlings infected via soil infestation than in the control seedlings. To our knowledge, this is the first pathogenicity test with H. annosum isolates and P. pinaster.     

Effect of pH,temperature and light on the pathogenicity of Cylindrocarpon destructans to pine seedlings in associative cultures with bacteria and actinomycetes1

The effect of pH, temperature and light on pathogenicity of two isolates of Cylindrocarpon destructans was studied. Both isolates were pathogenic to fir and pine seedlings. The degree of aggressiveness was affected by pH, temperature, light intensity and also by accompanying bacteria and actinomycetes.     

Pathogenicity of Malaysian Phytophthora palmivora on cocoa,durian, rubber and oil palm determines the threat of bud rot disease

The epidemic of bud rot disease affecting oil palm in Colombia is primarily caused by Phytophthora palmivora. The pathogen has a cosmopolitan presence that includes Southeast Asia, but to date, bud rot has not been reported in this region. This study provides an overview of the potential risk of Malaysian P. palmivora isolates cross‐infecting other host species, including cocoa, durian, rubber and Malaysian oil palm planting materials (Dura × Pisifera, D × P). On cocoa pods, the durian isolate PP7 caused dark brown necrotic lesions. Detached leaf bioassays showed that P. palmivora isolates PP3 and PP7 infected different hosts, except rubber foliage without wounding. Inoculation tests on cocoa, durian and rubber seedlings caused brown necrotic lesions when stems were wounded, with 10% mortality in cocoa and durian at 17 days post‐inoculation (dpi). However, no further infection was observed, and lesions closed within 14–28 dpi on the non‐wounded seedlings. Pathogenicity tests of oil palm seedlings inoculated with isolates PP3 and PP7 indicated that Malaysian P. palmivora isolates were not pathogenic to oil palms based on localized infection observed only through wounding. Overall, the work demonstrated that Malaysian P. palmivora isolates were able to cross‐infect multiple hosts but did not show severe infections on oil palms.     

Mating type ratios and pathogenicity in Diplodia shoot blight fungi populations: Comparative analysis

Diplodia sapinea and Diplodia scrobiculata are opportunistic pathogens of Pinus species. Several studies about taxonomy, impact and epidemiology of these fungi have been conducted in previous years, which have provided useful information and have raised new issues. These diseases produce a considerable impact on plantations resulting in significant economic losses. The main aims of this study are to increase the knowledge of the potential of genetic exchange and the relative aggressiveness of these organisms that can persist in healthy tissues of asymptomatic trees. A collection of 250 isolates among which are 149 strains collected from Pinus radiata plantations in Basque Country (Spain) and 101 strains from different countries was included in this work. Mating type ratios were analysed and compared using the structure of the MAT locus (MAT1‐1‐1 and MAT1‐2‐1). Inoculations of Pinus radiata seedlings were performed in a biosafety greenhouse (P2) to confirm pathogenicity of isolates and compare their aggressiveness. The frequency of occurrence of both idiomorphs of D. sapinea in Basque Country isolates was close to 1:1, however, for collection of isolates of this fungus from around the world, the ratio was 1:2. Furthermore, the spatial distribution of the two mating types in the Basque Country was random. Despite no detection of a sexual state, these results could suggest sexual reproduction behaviour. The pathogenicity of all strains in the collection was confirmed. Although aggressiveness (in terms of lesion lengths resulting from inoculation) varied greatly, no statistically significant effects of MAT type or pathogen species were detected.     

Pathogenic fungi associated with pre‐ and post‐emergence seedling blight of pine and cypress in Fars Province,Iran

Seed and root rot of pine and cypress seedlings cause heavy annual losses to forest nurseries in Fars Province. Root and crown samples of various species of conifers, such as Tehran pine (Pinus eldarica), brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazian cypress (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crown rot symptoms were collected from pine and cypress nurseries across Fars province at various time intervals. Infected tissues were washed and cultured on acidified potato dextrose agar (PDA) and corn meal agar (CMA) amended with Delvocide and ampicillin, with and without surface sterilization, respectively, and incubated at 25°C for 3–5 days. Decaying seeds of all plant species were also collected from nursery seed stocks and cultured on PDA. Hyphal tip isolates were used for further studies. Species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomatic seedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates of Phytophthora, Pythium and Rhizoctonia was confirmed on seedlings of all plant species, whereas Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pine and Arizona cypress. Virulent isolates identified as Phytophthora nicotiana var. parasitica, Pythium ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia‐like fungus caused root and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identified as Fusarium proliferatum also caused pre‐emergence seed decay of pine and cypress. Isolates of Phytophthora and Pythium were the most virulent of these fungal isolates.     

Sources of Diplodia pinea endophytic infections in Pinus patula and P. radiata seedlings in South Africa

Diplodia pinea, an opportunistic and latent pathogen, can significantly affect Pinus productivity worldwide. Despite being studied in South Africa for almost 100 years, the source of D. pinea inoculum responsible for seedling infection is unknown. The aim of this study was to determine the role of seed in vertical transmission of D. pinea and to investigate sources of inoculum leading to horizontal transmission to pine seedlings. Surface‐disinfected seeds were inoculated with spore and mycelium suspensions of D. pinea to determine its effect on germination. In addition, isolation of the fungus was performed from surface‐disinfected seeds, asymptomatic seedlings collected from nurseries, plantations where pines naturally regenerate and recently established fields, to assess transmission and incidence of endophytic D. pinea infections. Inoculation of seeds with D. pinea spore suspensions affected speed and rate of germination. The fungus was isolated from surface‐disinfected seeds in only a few instances (2–3%) and was not found in healthy seedlings collected from greenhouses and nurseries, suggesting that vertical transmission of the fungus does not occur or is rare. In contrast, D. pinea was isolated from 40% of seedlings obtained from the understory of mature P. patula trees showing that horizontal transmission from mature to young trees sustains the D. pinea inoculum in South African pine plantations.     

Uni- and binucleate Rhizoctonia spp. co-existing on the roots of Norway-spruce seedlings suffering from root dieback

Rhizoctonia fungi were isolated from the roots of 2-year-old nursery-grown Norway-spruce seedlings displaying root-dieback symptoms. The most frequently isolated species, a uninucleate Rhizoctonia sp., was found to co-exist with binucleate Rhizoctonia in the same root system of several seedlings. All the uninucleate isolates anastomosed with each other forming a single anastomosis group with common cultural characteristics. Binucleate Rhizoctonia isolates were divided into several, morphologically dissimilar anastomosis groups (AG-I, R. spp.). In a pathogenicity test under sterile conditions, isolates belonging to the uninucleate Rhizoctonia sp. infected all root regions, particularly the root tips, resulting in a stunted root-system morphology, as was also observed in the isolation material. Binucleate Rhizoctonia spp. colonized only basal root regions, occasionally infecting cortical cells with monilioid hyphae, and had no effect on root growth.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.