Association of seed‐borne Fusarium species on Pinus ponderosa with germination and seedling viability in Argentina

Pinus ponderosa seedlots from eight seed orchards in Western Patagonia, Argentina, were evaluated for the presence of Fusarium species, and isolates obtained were subjected to pathogenicity tests. The following species were isolated: Fusariumoxysporum, Fusarium equiseti, Fusarium verticillioides, Fusarium incarnatum, Fusarium acuminatum, Fusarium graminearum and Fusarium proliferatum. With the exception of F. proliferatum and F. graminearum all Fusarium species were recovered from P. ponderosa seedlings with symptoms of damping‐off. Five Fusarium species were re‐isolated from necrotic roots of seedlings showing chlorosis (F. oxysporum, F. equiseti, F. incarnatum, F. acuminatum and F. verticillioides). High damping‐off losses occurred in Fusarium‐contaminated seed of P. ponderosa and low disease incidence occurred on less contaminated seed. The role of seed‐borne inoculum in disease spread within forest nurseries is discussed.     

First report of Calonectria montana causing damping‐off disease on pine and spruce seedlings in Europe

In 2015–2016, damping‐off symptoms were observed on Pinus sylvestris and Picea abies seedlings in four forest nurseries in Poland. Some Calonectria isolates were obtained from symptomatic seedlings by culturing pieces of necrotic tissue on malt extract agar.Morphological and molecular analyses revealed that isolates represent Calonectria montana, a recently described species from natural forests in China. The isolates represent the first detection of this species outside of Asia. Pathogenicity tests demonstrated that C. montana is a severe pathogen of P. sylvestris seedlings.     

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.     

Pathogenicity of Fusarium verticillioides and Fusarium oxysporum on Pinus nigra seedlings in northwest Spain

Fusarium verticillioides may be responsible for causing significant damping-off damage similar to that incited by F. oxysporum on forest seedlings, resulting in considerable losses in nurseries in northwest of Spain. Traditionally, F. oxysporum has been considered the most important agent of this disease in Spanish forest nurseries. However, recent studies have showed that F. verticillioides also has been frequently isolated from diseased plants. This has increased the necessity for a more comprehensive knowledge of the behaviour and pathogenicity of both Fusarium spp. isolated from these sites. The effect of Fusarium spp. on seed germination and on seedling mortality was analysed by inoculating the fungus at seeding. The in vitro growth of the two species was studied and is discussed in relation to in vivo virulence. Both species caused a reduction in seed germination and an increase in seedling mortality. Mortality caused by F. verticillioides treatments occurred sooner than that for F. oxysporum and the growth rate of F. verticillioides was also greater.     

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.     

Fungi Isolated from Diseased Nursery Seedlings in Spain

Fungal endophytes in container-grown seedlings in forest nurseries located in the North Plateau of Spain have been studied. Influences of nursery, season (spring and autumn), host genus and plant part on those fungal assemblages have been also evaluated. Four nurseries were sampled in spring and three in autumn. Analysed material consisted of stems, roots and rhizosphere from five host species, Quercus ilex, Q. pyrenaica, Pinus pinea, P. nigra and P. sylvestris. A total of 581 isolates corresponding to 13 taxa were found in this study. Fusarium moniliforme, F. oxysporum and Trichoderma viride were the most frequently isolated fungi. Number of fungal species per observation differed significantly among the sampled parts. Multivariate Correspondence Analysis allowed the discrimination of samples in five groups based on nursery, season, host genus and plant part. Isolation frequencies of F. moniliforme and F. oxysporum were higher in the roots and in spring, whereas those of T. viride were higher in the rhizosphere and in autumn. A relationship was observed between presence of T. viride and absence of Fusarium spp. in forest nurseries. T. viride, could serve as a biological control agent against Fusarium spp. in forest nurseries.     

Seedborne Fusarium on Douglas-fir: Pathogenicity and seed stratification method to decrease Fusarium contamination

Twelve Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlots from coastal British Columbia were assayed for seedborne Fusarium. All of the seedlots were contaminated with Fusarium. Percent of nonstratified seeds from individual seedlots harboring Fusarium ranged from 0.3% to 95.4%. Sixty-seven percent of the seedlots had Fusarium on less than 2% of the seeds. Post-stratification seedborne Fusarium levels were significantly less for running water imbibition compared to standing water imbibition. However, seedling growth at a container nursery was not consistently different for stratified seed imbibed initially in standing or running water. Fusarium disease symptoms were not observed in the nursery environment. The species of Fusarium isolated from seed were F. acuminatum, F. avenaceum, F. lateritium, F. moniliforme, F. oxysporum, F. poae and F. sambucinum. Twelve Fusarium isolates, comprising six species, were assessed for pathogenicity. Disease symptoms were observed after four weeks incubation and Fusarium isolates ranged in virulence from low to high. Fusarium oxysporum isolates were the most pathogenic.     

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.     

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.     

Variation in virulence among isolates of Armillaria ostoyae

The virulence of Armillaria ostoyae isolates from coastal (16) and interior (33) British Columbia, elsewhere in North America (eight) and Europe (six) was assessed on 2‐year‐old Douglas‐fir seedlings in pots during a 3‐year trial. Isolates from most geographical locations infected similar proportions of seedlings, had similar average damage scores and killed a similar percentage of diseased seedlings. Isolates from the coastal region had a significantly higher probability than interior isolates that a diseased seedling received a damage score > 3 on a 1–5 scale, and coastal isolates killed a higher proportion of diseased seedlings than interior isolates. The mean damage score for isolates that had been in culture for 20–25 years was about 25% lower than that for recently collected isolates. The results indicate that the higher incidence and longer duration of mortality in the southern interior of British Columbia compared to the coast can not be attributed to greater virulence of interior isolates of A. ostoyae.     

The pine pathogen Diplodia sapinea is associated with the death of large Douglas fir trees

Climate change poses severe pressures to European conifer forests. Using non-native tree species, such as Douglas fir (Pseudotsuga menziesii), is one proposed strategy to circumvent adverse effects for forest management. However, novel forest health risks can impair the cultivation of non-native trees. In 2022, we observed large Douglas fir trees (approximately 40–50 years old, diameter at breast height (dbh) 21–41 cm) that had recently died in spring or summer 2022 in three forest stands in Eastern Austria. Intensive resin flow, blue-staining of the sapwood and the absence of bark- and wood-boring insects indicated a fungal infection. Isolations from blue-stained sapwood of the dead trees consistently yielded cultures of the opportunistic pathogen Diplodia sapinea. In a greenhouse wound inoculation experiment, seven D. sapinea isolates obtained from Douglas fir caused phloem necrosis, blue-staining of sapwood and mortality and thus displayed pathogenicity towards seedlings of both Ps. menziesii and its common host, Scots pine (Pinus sylvestris). Diplodia sapinea produced significantly longer areas of blue-stain as well as higher and faster mortality in Ps. menziesii compared to P. sylvestris. We conclude that D. sapinea substantially contributed to the death of seven of the 13 examined large Douglas fir trees. While this fungus has been described as a pathogen of young Douglas fir trees before, this is the first report that it can potentially kill large individuals of this conifer species under drought conditions. Thus, our results indicate that D. sapinea could represent a severe threat to the cultivation of Ps. menziesii in European forestry.     

Rhizomorph growth habit,saprophytic ability and virulence of 15 Armillaria species

The rhizomorph branching habit in soil, competitive saprophytic ability and virulence were determined for 15 species of Armillaria from Europe, North America and Australia and New Zealand. In soil, rhizomorphs of northern hemisphere species branched either monopodially or dichotomously, whereas all five species from Australia and New Zealand branched dichotomously. The dry weight of rhizomorphs produced in soil by isolates of a species and by species was very variable. Species with monopodially branched rhizomorphs had significantly higher saprophytic colonization scores than dichotomously branched species and scores were significantly higher in Garry oak than Douglas‐fir segments and in fresh than autoclaved segments. The damage to Douglas‐fir seedlings caused by isolates of most dichotomously branched species was significantly greater than that caused by monopodially branched species. Species producing dichotomously branched rhizomorphs were more aggressive than monopodially branched species, killing 80% (vs. 17%) of seedlings that died during the first year of the 2‐year experiment.     

The role of catechin and epicatechin in chemical defense against damping‐off fungi of current‐year Fagus crenata seedlings in natural forest

Current‐year Fagus crenata seedlings survive damping‐off in the forest‐edge plot compared to those in the closed‐stand plot. Greater illumination found in the forest‐edge plot is likely to influence the defense of seedlings against pathogenic attack, by increasing the amount of phenolic compounds and forming periderm. This study aimed to clarify the chemical defense system by (i) isolation of antifungal compounds from seedling hypocotyls in the forest‐edge plot, (ii) quantification of antifungal compounds in hypocotyls in the forest‐edge and closed‐stand plots, (iii) estimation of their antifungal activities against Cylindrocarpon sp. and Colletotrichum dematium and (iv) clarification of localization of phenolics in the hypocotyl tissues. Catechin and epicatechin were isolated as antifungal compounds showing inhibition of microconidia germination of Cylindrocarpon sp. and mycelial growth of Cylindrocarpon sp. and C. dematium. In the beginning of July when damping‐off was the most severe in the closed‐stand plot, the total amount of catechin and epicatechin in the forest‐edge seedlings was approximately three times greater than that in the closed‐stand seedlings. Flavan‐3‐ols, including catechin and epicatechin, were distributed mainly in the epidermal tissue on June 1 and in cortical tissues on June 15 and July 6, all from plants harvested from the forest‐edge plot. We conclude that F. crenata could survive damping‐off through chemical defense via catechin and epicatechin production and its distribution in seedling hypocotyls.     

Microbial communities in roots of Pinus sylvestris seedlings with damping‐off symptoms in two forest nurseries as determined by ITS1/2 rDNA sequencing

A methodological molecular procedure, which included extraction and cloning of the ITS1/2 rDNA of root‐associated organisms with subsequent transformation and sequencing of representative clones, was effective for detection, discrimination and determination of the frequency of the main damping‐off pathogens in roots of Pinus sylvestris seedlings growing in different forest‐tree nursery soils and exhibiting different rates of disease progress. Roots exhibiting slower damping‐off progression were colonized by Fusarium oxysporum, Neonectria radicicola (Ascomycota) and Pythium spp. (Oomycota), which comprised 50% of the microbial community. Roots exhibiting faster damping‐off progression were dominated by Thanatephorus cucumeris (Basidiomycota), which comprised 80% of the microbial community. The microbial community was more diverse in roots with slower damping‐off progression (14 species) than in roots with faster disease progression (seven species).     

Molecular phylogeny and pathotyping of Fusarium solani: a causal agent of Dalbergia sissoo decline

Sissoo (Dalbergia sissoo), commonly known as shisham, is amongst the finest woods of South Asia, but ‘wilt’ disease has caused a rapid decline in this species. The cause of the disease remains uncertain. The aim of this study is to identify the causal agent of the disease and characterize isolates made from diseased trees, based on genomic data and variations in virulence. Samples of infected roots, stems and the ooze exuded from infected trees were obtained from plants showing symptoms in different geographical regions of India for the isolation of microorganisms. Isolates were used to inoculate healthy plants. Based on the morphological characteristics, genus‐ and species‐specific PCR, and in silico analysis of 5.8S rDNA‐ITS regions, of the 38 fungal isolates, 24 and 14 were identified as Fusarium solani and Fusarium sp., respectively. In a pathotyping study, eighteen F. solani isolates, isolated from roots and stem parts of symptomatic plants, induced typical wilt symptoms when inoculated through soil and roots on D. sissoo seedlings of 1–15 months in age. The population of F. solani was the highest in infected roots and the lowest in parts of stems, gradually decreasing with height, and was isolated constantly up to approximately 40% height of the seedling. F. solani isolates used in inoculations were successfully re‐isolated from the rhizosphere, infected roots and wilted stems, as confirmed using isolate‐specific DNA fingerprints. Molecular phylogenies based on rDNA‐ITS sequences showed that the 38 isolates fell into 2 groups. Group I comprised of F. solani isolates from D. sissoo and F. solani sequences in the NCBI GenBank database, whereas group II included Fusarium isolates other than F. solani. These results are helpful in developing integrated control measures for this highly variable pathogen and to establish a base for future population studies.     

Microbial Inoculants for Sustainable Forests

Abstract

Fungal root pathogens are widespread and may cause substantial seedling losses in conifer nurseries. Furthermore, poor seedling survival and growth on reforestation sites results in reduced forest regeneration. Use of microbial inoculants for disease control and plant growth promotion has become an important endeavour. A microbial culture collection of 500 strains was assessed for biological control of fungal root pathogens and/or plant growth promotion of conifer seedlings. Seven of these strains showed significant suppres-sive effects on various soil-bome fungal pathogens. On Douglas fir, two strains, RAL3 and 64-3, reduced disease caused by Fusariumby. 7-42% in repeated growth room assays. The same strains significantly increased healthy stand of white spruce seedlings inoculated with Fusariumand Pythiumin a conifer nursery, and increased the survival of bare-root white spruce seedlings planted on a reforestation site by 19-23%. Both strains also significantly increased new root and total plant dry weights. Strain RAL3 in commercial formulation maintained a viable population of about log 8-9 cfu/ml for over a year when stored at 5°C. Strain survival on seed varied with conifer species. No decreases in bacterial populations were observed on seeds of jack pine or Douglas fir after 37 to 44 days storage at 5°C, but decreases were observed on seeds of white spruce and Scots pine. This study has provided candidate beneficial microbial inocu-lants which offer promise for development of commercial inoculants for the forestry industry.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

Fusarium root infection of container-grown Douglas-fir: effect on survival and growth of outplanted seedlings and persistence of the pathogen

Greenhouse-grown Douglas-fir (Pseudotsuga menziesii var.glauca [Beissn.] Franco) seedlings with roots infected with eitherFusarium oxysporum Schlect. orFusarium proliferatum (Matsushima) Nirenberg were outplanted on a forest site in northern Idaho, U.S.A. No residentFusarium populations were detected in the forest soil.Fusarium persisted the first four years on roots initiated during the greenhouse phase, but occurred sparingly or was absent on roots that grew after outplanting. Height growth was unaffected, and mortality was not often associated withFusarium. A seasonal pattern ofFusarium activity was observed. Low levels (10–40%) of initial root infection apparently have little adverse effect on outplanting performance of Douglas-fir seedlings.University of Idaho, Idaho Forest, Wildlife and Range Experiment Station Contribution No. 640.     

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.     

Virulence of Bursaphelenchus mucronatus to pine seedlings and trees under field cownditions

Bursaphelenchus mucronatus is a parasitic nematode of pine that is widely distributed in the natural pine forests of Asia and Europe. It has a very similar morphology and biology to that of Bursaphelenchus xylophilus, the causal agent of pine wilt disease, but has generally been considered to be non‐pathogenic to pine. However, in some provinces of China, B. mucronatus has been isolated from dead pine trees rather than B. xylophilus. Previous studies have shown that B. mucronatus can induce the death of pine seedlings under glasshouse conditions. To investigate the virulence of B. mucronatus, 2‐year‐old seedlings of Pinus massoniana and Pinus elliottii were inoculated with one of six isolates of B. mucronatus under field conditions in April 2014 and their condition was monitored over a year. The virulence of the six B. mucronatus isolates differed on the three host species: P. elliottii seedling mortality ranged from zero to six of the 18 inoculated seedlings, whereas P. massoniana seedling mortality ranged from four to 12 of the 18 inoculated seedlings. Three B. mucronatus isolates that appeared to cause different levels of mortality among the seedlings were used to inoculate 12‐year‐old Pinus thunbergii trees in August 2014. The trees were monitored for a year, during which time between 4 and 12 of the 18 inoculated trees in each treatment wilted and died. The average monthly temperature during the test period appeared to be similar to that of the historical average in the test areas; however, both study sites experienced above‐average rainfall. This study demonstrated that B. mucronatus has potential virulence on pine trees and provided experimental evidence that high temperatures or drought stress is not essential for the virulence of B. mucronatus.