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.     

Ectomycorrhizal inoculation of Douglas-fir transplanted container seedlings with commercially produced inoculum

Commercially produced vegetative inocula of Laccaria laccata and Hebeloma crustuliniforme successfully formed ectomycorrhizae with Douglas-fir transplanted container (plug+1) seedlings. After 4.5 months in containers, 83% and 90%, respectively, of short roots were mycorrhizal. L. laccata- or H. crustuliniforme-inoculated seedlings had significantly more mycorrhizal and total short roots than Pisolithus tinctorius-inoculated (4% mycorrhizal root tips) or uninoculated control seedlings. No significant differences were detected in seedling growth at the end of the container phase.After transplantation and growth in nursery beds for 17 months, mean new short root colonization of all seedlings was 80%. H. crustuliniforme persisted as a dominant mycorrhizal fungus on seedlings initially inoculated with this fungus. L. laccata-inoculated seedlings had 40% of their short roots colonized by L. laccata and another 40% by native fungi Rhizopogon and Thelephora spp. All mycorrhizae of control seedlings and those inoculated with P. tinctorius were formed by fungi native to the nusery beds. A significant fungal treatment effect was detected for shoot height only. Control seedlings were significantly taller than L. laccata-inoculated seedlings after transplanting.This article is part of senior author's thesis in partial fulfillment of the Ph.D. degree in the Department of Forest Science at Oregon State University, Corvallis.     

Effect of three species of bacteria on damping-off,root rot development,and ectomycorrhizal colonization of lodgepole pine and white spruce seedlings

Interactions between three species of bacteria (Burkholderia cepacia, Pseudomonas chlororaphis and Pseudomonas fluorescens), an ectomycorrhizal fungus (Paxillus involutus), and three root pathogenic fungi (Fusarium moniliforme, Fusarium oxysporum and Rhizoctonia solani) were studied. Burkholderia cepacia significantly reduced the in vitro mycelial growth of P. involutus, whereas, B. cepacia, Ps. chlororaphis, Ps. fluorescens and P. involutus reduced the mycelial growth of F. moniliforme, F. oxysporum and R. solani. Culture filtrates of B. cepacia, Ps. chlororaphis, Ps. fluorescens and P. involutus reduced conidial germination and increased the formation of chlamydospores of F. moniliforme and F. oxysporum. Burkholderia cepacia also reduced the formation of ectomycorrhizal short roots by P. involutus on lodgepole pine (Pinus contorta) and white spruce (Picea glauca) seedlings 2 months after inoculation. However, no significance difference in mycorrhizal short roots was observed after 4 months. The fungicide Anchor (a mixture of carboxine and thiram) significantly reduced root rot severity and increased the survival of lodgepole pine seedlings grown in a growth mix infested with F. moniliforme, F. oxysporum and R. solani. Control of the diseases of white spruce caused by these pathogens was not as successful. Treatment of seeds with either B. cepacia or P. involutus alone significantly increased the survival of seedlings grown in a mix that was inoculated with F. moniliforme and reduced the root rot severity caused by F. moniliforme and F. oxysporum, but not R. solani. Higher seedling survival and lower root rot severity were observed when conifer seeds were concomitantly inoculated with one of the bacterial species, P. involutus and Anchor.     

Responses of conifer species of the Great Lakes region of North America to inoculation with the pitch canker pathogen Fusarium circinatum

Five conifer species grown in the Great Lakes region of North America were examined for their susceptibility to Fusarium circinatum, (syns. Fusarium subglutinans f. sp. pini and F. moniliforme var. subglutinans), the causal agent of pitch canker. Three‐year‐old (3‐0) seedlings of red (Pinus resinosa), jack (P. banksiana) eastern white (P. strobus), Scots (P. sylvestris) and Austrian (P. nigra) pine were planted in 4 l pots in a greenhouse at Auburn University in November 1998. In April and June 1999, seedlings were inoculated by removing a needle fascicle approximately 5 cm from the terminal bud and placing a drop containing F. circinatum conidia on the wound. Resin production, canker length and seedling mortality were recorded 12 weeks later. Jack, Scots and eastern white pine were the most susceptible with Austrian and red pine more resistant to the fungus. F. circinatum was re‐isolated from 37% to 96% of inoculated seedlings. The susceptibility of jack, Scots and eastern white pine indicates a potential risk to these important species of the region if F. circinatum were to be introduced into the area.     

Influence of Microbial Inoculations on Cedrus deodara in Relation to Survival,Growth Promotion and Nutrient Uptake of Seedlings and General Soil Microflora

Abstract

Selected microorganisms (Laccaria laccata, Trichoderma viride, Bacillus subtilis, Pseudomonas corrugata and Azotobacter chroococcum) were used as inoculants to study their influence on seed germination, seedling survival and subsequent growth in Cedrus deodara. The microbial inoculations positively affected these parameters over control, in the following order: Azotobacter chroococcum Pseudomonas corrugata Bacillus subtilis Trichoderma viride Laccaria laccata.The major cause of seedling mortality was found to be (1) wilting of emerged seedlings by Fusarium oxysporum, and (2) cutworm attack. Bacterial inoculations were found to be superior in suppressing the proliferation of Fusariumsp., thereby enhancing seedling survival. One of the bacterial inoculants, B. subtilis, resulted in 76% seed germination, compared to 54% in control. Though inoculation with L. laccataresulted in maximum mycorrhizal infection (up to 72%) compared to48%in control seedlings, it afforded least protection against Fusariumsp., as well as minimal growth promotion. On the other hand, inoculation with T. viridesignificantly controlled wilting of seedlings and improved plant growth. Microbial inoculations were found to influence the soil microflora, nutrient status of the rhizosphere soil and that of different parts of seedlings. Enrichment of carbon was recorded in the rhizosphere soil. Enhanced nitrogen, phosphorus and potassium content of various plant parts indicated a positive influence also on nutrient uptake. The present study has implications for large scale forestry programs.     

Etiology of oak decline in Spain

In different areas of Extremadura, Western Spain, soil samples were taken at the bottom of holm oak (Quercus ilex) trees that were showing decline symptoms. Half of each sample was sterilized, and acorns were sown in both sterilized and nonsterilized soil samples. The resulting seedlings were used as baits for the isolation of fungi. Seedlings growing on the natural, nonsterilized substrate were characterized by having a lower vegetative growth than the ones growing on the sterilized soil samples, and most of them died. Phytophthora dnnamomi was consistently isolated from their roots. Fusarium oxysporum was also isolated as well as different species of Pythium, although to a lesser extent. Pathogenicity tests were performed on holm oak seedlings with five different isolates of P. chinnamomi, with F. oxysporum, Pythium and with a mixture of the three fungi. All the inoculated seedlings with P. chinnamomi developed root rot and grew slowly, and 35.7% of them died up to the end of the experiments. P. chinnamomi was consistently isolated from their roots, indicating that this fungus is the causal agent of holm oak decline. However, F. oxysporum caused similar symptoms on oak seedlings as P. chinnamomi, and was isolated also from the roots, although its frequency was lower than that of. P. cinnamomi.     

Some factors affecting canker formation in American beech inoculated with Nectria coccinea var. faginata1

Combinations of Nectria coccinea var. farinata (the primary causal agent of beech bark disease), Fusarium oxysporum, and associated bacteria were inoculated into American beech trees at two sites in New Hampshire. Neither F. oxysporum nor bacteria appeared to affect the size of cankers formed. Canker size was significantly related to bark thickness at one site when only one mating type of the pathogen was inoculated alone.     

Epiphytology of Scleroderris lagerbergii in a kettle frost pocket

Both the fungus Scleroderris lagerbergii Gremmen and summer frost were implicated in the death of seedlings of Pinus resinosa Ait. and Pinus banksiana Lamb. [=P. divaricata (Ait.) Dumont] in the Goulais Kettle Frost Pocket in central Ontario, Canada. This fungus was responsible for the preponderance of early seedling mortality, with the survival differential between inoculated and uninoculated seedlings decreasing slightly with increasing elevation in the depression. A mechanism whereby inoculum is recirculatcd to the seedlings by wind vortices was identified and described.     

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.     

Resistance of Pinus contorta to the European race of Gremmeniella abietina

Pinus contorta seedlings, together with Pinus resinosa and Pinus banksiana seedlings, were planted adjacent to 25‐year‐old red pine trees infected by the European (EU) race of Gremmeniella abietina. Resistance to this race was assessed over 5 years. All P. resinosa seedlings were dead after that period while 65% of P. contorta and 86% of P. banksiana seedlings appeared resistant to the disease. The tip blight that occurred on P. contorta was slightly longer than that observed on P. banksiana. In microscopy, one, two, or even more suberized boundaries were seen to be initiated near the surface of the shoot at the base of healthy needles where they extended downward in the direction of the vascular cambium. Suberized boundaries occasionally crossed the xylem and joined together in the pith region to form continuous barriers around necrotic tissues. However, in most cases, these suberized barriers were not continuous across the shoot and compartmentalization was then completed by other barriers mainly constituted of parenchyma cells and xylem tracheids that accumulated phenolic compounds. Meristematic‐like cells were observed adjacent to the necrophylactic periderm. Tissue regeneration, restoration of cambial activities and formation of traumatic resin canals also seemed to be associated with the defence system of P. contorta against the EU race of G. abietina.     

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.     

Influence of the fertilisation method in controlled ectomycorrhizal inoculation of two Mediterranean pines

The influence of the fertilisation method: soluble (SF) vs. slow-release fertiliser (SRF) and of inoculation with Laccaria laccata (Scop.) Fr., Pisolithus tinctorius (Pers.) Coker & Couch and Melanogaster ambiguus (Vittad.) Tul & C. Tul. on ectomycorrhizal colonization and growth of Pinus pinea L. and Pinus pinaster Ait. was evaluated. For both pines, mycorrhization with L. laccata was not affected by the fertilisation method. Percentages of ectomycorrhizas (ECM) formed by P. tinctorius were dependent on the fertilisation method, the inoculum type (vegetative or spores) and the pine species involved. ECM formed by M. ambiguus were increased with fertilisation in both pines. Inoculation significantly improved P. pinea biomass when seedlings were fertilised with SRF whereas no effect was found in non-fertilised ones. For non-fertilised P. pinaster, inoculation with L. laccata and both inocula of P. tinctorius increased seedling biomass whereas fertilisation neutralised the fungal effect. Fertilisation increased P. pinea and P. pinaster biomass, independently of the inoculation treatment.     

Growth stimulation of young oak plantations inoculated with the ectomycorrhizal fungus Paxillus involutus with special reference to summer drought

Nursery-grown Quercus petraea and Q. robur seedlings were inoculated with the ectomycorrhizal fungi Paxillus involutus, Hebeloma crustuliniforme or Laccaria laccata. At the end of the first growing season, the seedlings with roots colonized with the inoculated fungi were outplanted at two sites in northeastern France in 1985 or 1988. P. involutus was found to be the most competitive and efficient of the three fungi tested; its mycorrhizas were present 7 yrs after outplanting and it markedly improved the growth of both oak species. In the case of Q. robur, the growth stimulation was more marked on years with a dry summer. These results are discussed in terms of water relations and of competitiveness of the introduced versus resident symbiotic fungi.     

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.     

Pathogenicity of A and B morphotypes of Sphaeropsis sapinea confirmed on American larch (tamarack) and European larch

Fungi identified as Sphaeropsis sapinea (or synonyms) have been reported from Larix. Lack of pathogenicity tests, confusion about the identity of isolates mentioned in previous literature, and existence of distinct populations (A and B morphotypes) of the pathogen indicated the need to evaluate the ability of S. sapinea to cause disease of larch. Elongating snoot tips of Larix decidua, Larix laricina, and known hosts of the pathogen, Pinus banksiana and Pinus resinosa, were inoculated with water-agar plugs colonized with an A or a B isolate, or sterile plugs (controls). Each of five replications included five seedlings for each treatment-species combination. After 6 weeks, no symptoms had developed on control seedlings, but two-way analyses of variance revealed significant effects of isolate morpnotype and host on both incidence and severity of disease (values of p ≤ 0.01). The A isolate killed almost all shoot tips, but the B isolate killed from no P. resinosa shoots to 56% of L. laricina shoots. The average length of shoot killed by the A isolate was also greater than that killed by the B isolate. Response to the B isolate again varied among species, with greater average lengths of shoot killed on the larches (compared with the pines). Both A and B morphotypes of 5. sapinea should be considered among the fungi encountered on L. decidua and L. laricina.     

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.     

Effects of Acidic Precipitation and Ectomycorrhizal Inoculation on Growth,Mineral Nutrition,and Xylem Water Potential of Juvenile Loblolly Pine and White Oak

Abstract

Individual and interactive effects of simulated acidic rainfall and mycorrhizal inoculation on growth and nutrient and water relations of loblolly pine (Pinus taedaL.) and white oak (Quercus albaL.) grown in a loam soil were examined. Seedlings of each species inoculated with basidiospores of the ectomycorrhizal fungus Pisolithus tinctorius(Pers.) Coker and Couch, a known my-cobiont of both loblolly pine and white oak, and uninoculated control seedlings received two simulated rains per week of either pH 3.6, 4.2, or 4.8 for 26 weeks. Higher acidity rainfall reduced the growth but increased mycorrhizal colonization of loblolly pine, while both loblolly pine and white oak exposed to these rains exhibited greater foliar injury. Inoculation with P. tinctoriusincreased growth and reduced foliar injury of both species. Foliar concentrations of P, S, and Cu in loblolly pine and white oak, Ca in loblolly pine, and Fe and Zn in white oak decreased with increasing rain acidity while the Al concentration of both species increased. Higher rainfall acidity also reduced soil pH and Ca and Mg concentrations while increasing soil AI. Foliage of inoculated seedlings of both species had higher N and P concentrations and lower Al concentrations than control seedlings. Following the final rain applications, a drought cycle was simulated by withholding irrigation for two weeks during which seedling xylem pressure potential and soil water potential were measured. One day after cessation of irrigation, xylem pressure potential of loblolly pine that had received pH 3.6 rains was lower than that of other treatments. Thereafter, xylem pressure potential and soil water potential of the inoculated treatment decreased below those of the control treatment in both species. These results suggest that acid deposition is detrimental to juvenile loblolly pine and white oak, but the magnitude of this effect is less than the positive response to ectomycorrhizal inoculation.     

The influence of Desert False Indigo, <Emphasis Type="Italic">Amorpha fruticosa</Emphasis>, and three arbuscular mycorrhizae fungi on the growth of <Emphasis Type="Italic">Populus ussuriensis</Emphasis> seedlings

The influence of Desert False Indigo, Amorpha fruticosa, on the growth of Populus ussuriensis seedlings inoculated with three arbuscular mycorrhizae (AM) fungi (Glomus mosseae, Glomus intraradices, Glomus sinuosa) was studied using the nylon net method. The results showed that all three AM fungi infected P. ussuriensis seedlings and G. intraradices and also G. mosseae infected A. fruticosa. The AM fungi promoted growth of P. ussuriensis and Desert False indigo seedlings. Moreover, under co-cultivation with A. fruticosa, the biomass of P. ussuriensis increased significantly. The concentration of nitrogen in P. ussuriensis grown with A. fruticosa and the concentration of soluble nitrogen in the rhizosphere were also higher than when grown alone. Hypha were found on the two plant seedlings inoculated with G. mosseae and G. intraradices, suggesting that AM fungi may transport nutrients from seedlings of A. fruticosa to the rhizosphere of P. ussuriensis seedlings, which may have promoted the growth of P. ussuriensis. The AM fungi played a critical role on the effect of A. fruticosa on growth of P. ussuriensis.     

Silvicultural options to promote seedling establishment on Kalmia–Vaccinium-dominated sites

Seedling growth is often hampered on sites dominated by Kalmia angustifolia. In June 2000, a trial was established on a clear-cut site in Quebec, Canada, with a high cover of Kalmia and Vaccinium species. The objectives were to evaluate how soil scarification and fertilization at the time of planting influence early growth and establishment of black spruce [Picea mariana (Mill.) BSP] and jack pine (Pinus banksiana Lamb.) seedlings. During the first 2 years, scarification reduced Kalmia cover three-fold and doubled the distance from seedlings to the nearest Kalmia stem. Scarification did not increase soil-extractable NH₄-N concentration, and reduced soil potassium, calcium and magnesium. Scarification had no effect on seedling water stress. Seedling growth improved and foliar nutrient concentrations were generally higher in scarified plots than in unscarified control plots. No differences were observed between single- and double-pass scarification for any variables except for ground-level stem diameter of seedlings, which was greater with double-pass scarification (12.1 vs 13.1 mm). Spot fertilization increased seedling growth and foliar nitrogen concentrations. Jack pine growth was greater than black spruce growth, an effect enhanced when seedlings were fertilized.     

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.