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.     

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.     

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.     

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.     

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.     

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.     

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.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.     

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.     

Rhizoctonia solani AG 2-1 as a causative agent of cotyledon rot on European beech (Fagus sylvatica)

Rhizoctonia solani was frequently isolated in the Italian Alps from nursery-grown European beech (Fagus sylvatica) seedlings displaying symptoms of cotyledon rot. Koch's postulates were verified and mode of infection of the associated isolates was investigated with light and scanning electron microscopy. Population structure of the pathogen was investigated by scoring the anastomosis reaction type in pairings between different isolates from the same seedbed. One pathogen genotype showed a large distribution area within the seedbed, this implying that the inoculum had been building up in the seedbed over a longer time period. Hyphal anastomosis tests and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA indicated that the pathogen belongs to AG 2-1 of R. solani. ITS sequence analysis indicates that the isolates from beech are closely related to R. solani isolates causing a disease on tulip. The striking similarities between the two diseases are discussed.     

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.     

Incidence and phylogenetic analyses of Armillaria spp. associated with root disease in peach orchards in the State of Mexico,Mexico

Incidence of peach [Prunus persica (L.) Batsch] tree mortality attributed to Armillaria root disease was assessed from 2009 to 2011 in 15 orchards in the State of Mexico, Mexico. Incidence increased gradually every year of assessment, reaching average values of 9.7, 15.3 and 20.3% tree mortality and 23.2, 24.7 and 28.3% disease‐impacted area of the orchards during 2009, 2010 and 2011, respectively. The cultivars ‘Nemaguard’ and ‘Criollo of La Goleta’, a local rootstock used in the region, were both susceptible to the disease. To identify species of Armillaria isolated from infected peach trees, two nuclear rDNA regions (partial 5.8S‐ITS2‐LSU D‐domains and partial 3′ LSU‐IGS1) and the translation elongation factor‐1α (tef‐1α) gene were sequenced and compared with sequences of known Armillaria species. DNA sequence analysis from 49 Armillaria isolates revealed that five isolates (10.2%) were Armillaria mellea and eight isolates (16.3%) were Armillaria gallica. DNA sequences from the remaining 36 isolates (73.5%) showed no close similarity to Armillaria sequences in GenBank, and apparently represent an undescribed Armillaria species. This undescribed species was the most widely distributed in the region of study. Separate phylogenetic analyses of the LSU region (D1–D3 domains concatenated with the partial 3′ end) and the tef‐1α region show that the undescribed species is quite distinct from other Armillaria spp. reported in North America.     

Identification of the Armillaria root rot pathogen in Ethiopian plantations

Armillaria root rot is a well‐known disease on a wide range of plants, world‐wide. In Ethiopia, the disease has previously been reported on Pinus spp., Coffea arabica and on various native hardwoods. The causal agent of the disease has been attributed to Armillaria mellea, a species now known to represent a complex of many different taxa. The aim of this study was to determine the extent of Armillaria root rot and the identity of the Armillaria sp. in Ethiopian plantations. As part of a plantation disease survey in 2000 and 2001, samples were collected in plantations at and around Munessa Shashemene, Wondo Genet, Jima, Mizan and Bedele, in south and south‐western Ethiopia. Basidiocarps were collected and their morphology studied. Morphological identification was confirmed by sequencing the intergenic spacer (IGS‐1) region of the ribosomal rRNA operon and comparing data with published sequences of Armillaria spp. Armillaria isolates were collected from Acacia abyssinica, Pinus patula, Cedrela odorata and Cordia alliodora trees. Sporocarps were found on stumps of native Juniperus excelsa. Basidiocarp morphology and sequence data suggested that the fungus in Ethiopia is similar to that causing disease of Pinus spp. in South Africa and previously identified as A. fuscipes. This identification was confirmed for all isolates, based on sequence data. Armillaria fuscipes is known to be common in southern Africa. Its widespread occurrence in Ethiopia suggests that it is also the major cause of Armillaria root rot in that country.     

Isolation and identification of endophytic bacteria with plant growth promoting and biocontrol potential from oak trees

Samples of roots, leaves and stems of healthy oak trees were collected from various locations in the Baneh and Marivan regions, Iran. In total, 63 endophytic bacteria were isolated and grouped according to phenotypic properties. Seven selected isolates were further identified by partial sequencing of the 16S rRNA gene. Isolates Pp54, Pp88, Pp95 and Pp177 were Pseudomonas spp., isolates Sm59 and Sm79 were Stenotrophomonas spp., and isolate Bf 172 was a Bacillus sp. The ability of these isolates for plant hormone production such as auxin and gibberellin was evaluated, along with siderophore production, phosphate solubilization, atmospheric nitrogen fixation, protease and hydrogen cyanide production. All strains produced auxin and gibberellin in different amounts. Atmospheric nitrogen fixation ability was positive for strains Sm79, Pp54, Pp88 and Pp95. All strains except Sm79 solubilized phosphate. Strains Sm59, SM79, Pp88, Pp177 and Bf172 produced protease. Pp88, Pp95, Bf172 and Pp177 were able to produce siderophore. Strains Sm79 and Pp95 released low concentrations of hydrogen cyanide. Amongst the strains tested, Pp95, Pp88, Pp177 and Sm79 had different inhibitory effects on the bacterial plant pathogen, Pseudomonas syringae pv. syringae under in vitro conditions. This is the first reported case of endophytic Bacillus, Pseudomonas and Stenotrophomonas strains from oak trees in Iran.     

Effects of endophytic and ectomycorrhizal basidiomycetes on Quercus virginiana seedling growth and nutrient absorption

Quercus virginiana (Fagaceae), native to the southeastern United States, has recently been introduced to the Yangtze Delta (China) and is often planted in landscaping and in coastal shelter forests. Here, we performed pot inoculation assays to evaluate the effects of two types of basidiomycetous root fungal symbionts, across four orders, on the growth and nutrient absorption of Q. virginiana seedlings. The results indicated that two isolates showed plant growth-promoting activities 2 months after inoculation. In particular, the ectomycorrhizal Sistotrema sp. (Cantharellales) had a pronounced effect on root development and morphology. With regard to nutrient absorption, the total nitrogen content in aboveground tissues was improved in inoculated seedlings, although most differences were not significant. Meanwhile, the total phosphorus contents in seedlings were either positively or negatively affected, probably depending on the symbiont type. Interestingly, the total potassium content in stems and leaves was significantly enhanced in all treatment groups. In addition, indole-3-acetic acid and abscisic acid production were determined in Sistotrema sp. and Atractiella rhizophila (Pucciniomycotina) by high-performance liquid chromatography tandem mass spectrometry. Our findings suggest that both endophytic and ectomycorrhizal fungi have beneficial effects on Q. virginiana growth, and highlight the potential for the development of mycobiome-based seedling raising techniques.     

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.     

A single dominant Ganoderma species is responsible for root rot of Acacia mangium and Eucalyptus in Sumatra

Ganoderma root rot is the most serious disease affecting commercially planted Acacia mangium in plantations in Indonesia. Numerous Ganoderma spp. have been recorded from diseased trees of this species and to a lesser extent Eucalyptus, causing confusion regarding the primary cause of the disease. In this study, a large collection of Ganoderma isolates were obtained from the roots of A. mangium showing early signs of root rot in disease centres in South Sumatra plantations. Isolates were also collected from Eucalyptus roots at Lake Toba in North Sumatra showing similar symptoms as well as from sporocarps connected to these samples. Phylogenetic analyses showed that a single Ganoderma sp., identified as G. philippii, is the major causal agent of Ganoderma root rot on A. mangium. Results from this study also showed that the isolates obtained for Eucalyptus trees in North Sumatra belong to G. philippii. Isolates from roots and connected fruiting bodies together with the morphology of the fruiting structures confirmed this identification. Symptoms associated with this pathogen are obvious and it should not be confused with other diseases. Other Ganoderma spp. found in disease centres are considered to be of minor importance and management strategies for root rot should be focused on G. philippii.     

Chrysoporthe puriensis causing canker and mortality in Pleroma mutabile in the Atlantic Forest,Brazil

Fungi of the Cryphonectriaceae family are globally known to be tree pathogens. In Brazil, several species of Chrysoporthe have been found causing stem and branch cankers in Pleroma (= Tibouchina) spp. Recently, Chrysoporthe puriensis was described as a new species causing stem and branch cankers in Pleroma granulosum, Pleroma candolleanum, and Pleroma heteromallum, all native species of the Melastomataceae family. During an investigation to collect isolates of the Cryphonectriaceae family in an important Brazilian biome, the Atlantic Forest, in Serra do Mar, structures typical of Chrysoporthe species were found in a different host, Pleroma mutabile. Fruiting bodies present in the bark of these trees were collected and isolated. The isolates were submitted on morphological characterization and phylogenetic analyses of the internal transcribed spacer (ITS) and β-tubulin gene regions using Maximum likelihood, Bayesian inference and Maximum parsimony methods. The isolates collected, for this study, were identified as C. puriensis. Pathogenicity tests on seedlings of Pleroma species and Eucalyptus clones revealed C. puriensis can infect and cause canker in these plant species as mortality. The results demonstrate the importance of delimiting the C. puriensis collection range to track its dissemination in other hosts. No host specificity was observed in the inoculation tests, suggesting this is an important finding, the pathogen causes diseases and mortality in several plants of the Atlantic Forest. Additionally, the pathogen can affect others hosts, such as Eucalyptus clones in commercial plantations.     

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.