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.     

Gliocladium virens in an alginate prill ineffective as a biological control of Fusarium root disease in container-grown Douglas-fir

An alginate prill formulation of Gliocladium virens (GL-21) was added as a top-dressings (54 g per m²) or incorporated into medium (1.2 kg per m³) used to grow Douglas-fir seedlings in styrofoam containers. Seedlings in the top-dress treatment were similar to control seedlings; infection and colonization by naturally-occurring Fusarium was unaffected by treatment. Incorporated G. virens reduced seedling growth and increased occurrence and colonization intensity of Fusarium. In a laboratory experiment, inoculating Douglas-fir seedlings with G. virens (10% w/w) prior to inoculation with Fusarium increased survival time when compared to concurrent inoculations of fungi.University of Idaho, Idaho Forest, Wildlife and Range Experiment Station Contribution No. 742.     

Influence of Microbial Inoculants on Seedling Production in Teak (Tectona grandis L.f.)

Abstract

An experiment was conducted in forest college campus, Mettupalayam, India with an objective of studying the influence of microbial inoculants on seedling growth of teak. The fruits were collected from the seed production area in top slip (Tamil Nadu) from 63-year mother trees. The microbial inoculants viz., Azospirillum, Azotobacter, phosphobacteria, AMF, Azospirillum + AMF, Azotobacter+ AMF, phosphobacteria + AMF and an uninoculated control formed the treatments. The inoculants were mixed sand and red soil at 1:1:3 ratio, respectively, for preparing nursery mixture for filling the poly pots. The seeds were sown into the poly pots @ two seeds per pot. The experiment was set up in a randomized Block Design with three replications. Biometric observations on various growth attributes were recorded six months after treatment and also their survival in the main field-tested for one year. Azotobacter gave best performance with respect to shoot length, shoot weight and leaf area and combined inoculation of phosphobacteria and AMF gave higher root length and biomass, dry matter production and fine root weight. The survival was better in seedlings that were inoculated with phosphobacteria and AMF. However, below ground biomass and out planting survival during early phase are critical in any tree species. Hence it is concluded that inoculation of phosphobacteria and AMF conjointly be done for better survival and growth of teak seedlings.     

Pathogenicity of Bursaphelenchus sexdentati,Bursaphelenchus leoni and Bursaphelenchus hellenicus on European pine seedlings

In Greece extensive pine wilting is associated with the presence of nematodes of the genus Bursaphelenchus. In order to check the pathogenicity of Bursaphelenchus sexdentati, Bursaphelenchus leoni and Bursaphelenchus hellenicus, inoculation tests were carried out on 3‐year‐old seedlings of Pinus brutia, Pinus halepensis, Pinus nigra, Pinus pinaster and Pinus sylvestris. For inoculations, 6000 nematodes in 0.5 ml of water were used per seedling. Bursaphelenchus sexdentati proved to be highly virulent causing mortality of up to 100%, followed by B. leoni whereas B. hellenicus was nonpathogenic.     

In vitro ectomycorrhizal formation of Pinus patula, P. pseudostrobus, P. oocarpa and P. elliotii grown in southern India

In vitro syntheses of ectomycorrhizas of Pinus patula, P. pseudostrobus, P. oocarpa and P. elliotii with Amanita muscaria, Laccaria fraterna, L. laccata, Pisolithus tinctorius, Rhizopogon luteolus, Scleroderma citrinum and Thelephora terrestris were attempted. Most of the fungi initiated mycorrhizal formation within 2 weeks after inoculation. Laccaria fraterna and S. citrinum formed mycorrhizas after 6 weeks of inoculation. Amanita muscaria failed to form mycorrhizas during the 12 week period of observation. The degree of colonization of different mycorrhizal fungi varied with different species of Pinus.     

Growth response and nutrient utilization of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> seedlings inoculated with bioinoculants under tropical nursery conditions

We investigated the role of tetrapartite associations between an arbuscular mycorrhizal (AM) fungus (Glomus geosporum), phosphate solubilizing bacteria (Paenibacillus polymyxa), Frankia and Casuarina equisetifolia on growth, nutrient acquisition, nutrient utilization and seedling quality of C. equisetifolia. Seedlings of C. equisetifolia were grown in an Alfisol soil and inoculated with G. geosporum, P. polymyxa and Frankia either individually or in combinations. Inoculation of bioinoculants stimulated seedling growth, the efficiency of nutrient uptake and improved seedling quality. However, microbial inoculation generally reduced the efficiency of nutrient utilization in dry matter production (nutrient use efficiency). Inoculation of P. polymyxa or Frankia increased the extent of AM colonization, which resulted in the accumulation of the nutrients. Seedlings inoculated with Frankia and G. geosporum had more, and heavier nodules compared to seedlings inoculated with Frankia alone. Dual inoculation of microbes was more effective than individual inoculations. The growth response of seedlings to inoculation involving all the microbes was greater than the response to either individual or dual inoculations. The results of this study showed that the tetrapartite association could improve the growth, nutrient acquisition and seedling quality of C. equisetifolia under tropical nursery conditions.     

Inoculation of ectomycorrhizal fungi contributes to the survival of tree seedlings in a copper mine tailing

To advance our understanding of the effects of inoculation with ectomycorrhizal fungi (EMF) on seedling colonization in mine wastelands, we conducted a field experiment in a copper tailing. Six-month-old seedlings of Japanese red pine (Pinus densiflora) and oak (Quercus variabilis) separately inoculated with three EMF species (Pisolithus sp., Cenococcum geophilum, Laccaria laccata) were transplanted to the copper tailing. The survival rates of tree seedlings were monitored monthly, and growth (biomass and height), contents of nutrients and heavy metals (K, P, Ca, Mg, Cu, Zn), and mycorrhizal infection rates of seedlings were determined 6 months after planting. Oak seedlings exhibited higher survival rates than pine seedlings after 6 months of growth on the tailing. EMF inoculations of pine seedlings significantly enhanced their survival, growth, and nutrient uptake. In contrast, EMF inoculations of oak seedlings improved growth only in terms of biomass. Additionally, EMF inoculation caused pine seedlings to accumulate more Cu and Zn in roots compared to non-inoculated seedlings, whereas inoculation inhibited the accumulation of heavy metals in shoots. However, similar results were not observed in oak seedlings. Observations of roots indicated that the rates of mycorrhizal infection of both tree species had dramatically declined at harvest time. In conclusion, ectomycorrhizal symbioses can improve the survival and performance of pine seedlings in mine tailings. The present study provided direct evidence of the importance of EMF inoculation of seedlings to the reforestation of mine wastelands.     

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.     

Fusarium spp. and Pinus strobus seedlings: root disease pathogens and taxa associated with seed

Eastern white pine (Pinus strobus L.) seeds weresown in soil infested with Fusarium proliferatum, rootnecrosis developed on seedling roots, and F. proliferatumwas reisolated from symptomatic roots; thus, demonstrating thatF. proliferatum is pathogenic toeastern white pine seedlings. Soils infested with F.acuminatum or F. sporotrichioides resulted infew diseased seedlings. Seedlings with root rot generally showed reductions inseedling height. All Fusarium species tested wererecoveredfrom rhizosphere soil samples. Three seedlots of Pinusstrobus were examined for Fusarium infestation.Fusarium species were recovered from most seeds in twoseedlots. Fusarium proliferatum and F.sporotrichioides were the predominant species isolated. Additionalspecies not previously reported from P. strobus included:F. acuminatum, F. chlamydosporum,F. equiseti, F. graminearum,F. moniliforme, F. poae, F.polyphialidicum, F. heterosporum, F.sambucinum, and F. semitectum.     

Growth response of <Emphasis Type="Italic">Acacia mangium</Emphasis> Willd. seedlings to arbuscular mycorrhizal fungi and four isolates of the ectomycorrhizal fungus <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis> (Pers.) Coker and Couch

Some Acacia mangium Willd. plantations in Asia grow poorly due to low soil fertility and the absence of compatible mycorrhizal fungi. This legume tree can be colonized by arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi but inoculation is not routinely practiced. This study aimed to compare the effectiveness of AM fungi and four isolates of the ECM fungus Pisolithus tinctorius (Pers.) Coker and Couch (Pt) in promoting growth of A. mangium seedlings under glasshouse conditions. AM inoculants were: Glomus etunicatum, G. fasciculatum, G. macrocarpum and Gigaspora margarita and mixed species extracted from rhizosphere soil of a Populus stand in Suwon (AMKFRI), and Carex (AMM6) and Populus (AMM7) growing in mine tailings in Korea. Pisolithus isolates were from Philippines (PtPhil) and Korea (PtKFRI, PtMKACC, PtKACC). Generally, ECM fungi promoted height and diameter growth of A. mangium more than the AM inoculants. The Korean Pisolithus increased plant dry weight by 122–145%, mixed AM inoculants by 61–97%, and Glomus and Gigaspora by 45–72% over the control. PtKACC gave the highest root colonization and promoted the highest growth and concentration of most nutrients. Mycorrhizal root colonization was positively correlated with plant dry weight, Na, Fe and Cu concentrations and N, P, K, Ca, Na, Fe and Cu contents. In conclusion, the results provide strong evidence for benefits of mycorrhizal inoculation on A. mangium seedlings under glasshouse conditions. The Korean Pisolithus isolates (particularly PtKACC), and two AM fungi (AMKFRI and AMM6) are potential mycorrhizal inoculants but their effectiveness and persistence should be determined on degraded lands in tropical countries where A. mangium is being planted for rehabilitation.     

Effect of the arbuscular mycorrhizae <Emphasis Type="Italic">Glomus fasciculatum</Emphasis> and <Emphasis Type="Italic">G. macrocarpum</Emphasis> on the growth and nutrient content of <Emphasis Type="Italic">Cassia siamea</Emphasis> in a semi-arid Indian wasteland soil

The effect of two arbuscular mycorrhizal (AM) fungi, Glomus fasciculatum and G. macrocarpum on shoot and root dry weights and nutrient content of Cassia siamea in a semi-arid wasteland soil was evaluated. Under nursery conditions, mycorrhizal inoculation improved growth of seedlings. Root and shoot dry weights were higher in mycorrhizal than non-mycorrhizal plants. The concentration of P, K, Cu, Zn and Na was significantly higher in AM inoculated seedlings than in non-inoculated seedlings. Mycorrhization led to decrease in alkalinity of the rhizosphere soil from pH 8.5 to 7.4. Under nursery conditions, the degree of mycorrhizal dependency increased with age of C. siamea seedling. On transplantation to the field, the survival rate of mycorrhizal seedlings (75%–90%) was higher than that of non-mycorrhizal seedlings (40%). Besides better survival rate, AM inoculation improved the growth performance of seedlings in terms of height and stem diameter. Among the two AM fungi used, the efficiency of Glomus macrocarpum was higher than that of G. fasciculatum under both nursery and field conditions.This revised version was published online in May 2005 with corrected page numbers.     

香荚兰根腐病主要病原的生防因子作用比较

尖镰孢香荚兰专化型是香荚兰根腐病的主要病原。本实验用木霉,枯草芽孢杆菌,荧光假单孢菌对尖镰孢菌香荚兰专化型进行实验室的拮抗作用测定,并进行了比较。结果表明:木霉对尖镰孢香荚兰专化型的的抑制作用最强,抑制率可达78.57%,枯草芽孢杆菌对尖镰孢香荚兰专化型的的抑制作用较强,抑制率可达78.12%,而荧光假单孢菌Ph002对该菌抑制效果则很弱,最高才有39.13%。总之,三种生防菌的抑制效果是:木霉>枯草芽孢杆菌>荧光假单孢菌。     

Influence of an indigenous European alder (Alnus glutinosa (L.) Gaertn) rhizobacterium (Bacillus pumilus) on the growth of alder and its rhizosphere microbial community structure in two soils

Europeanalder seedlings were inoculated with a suspension of the putative plant growthpromoting rhizobacterium (PGPR) Bacillus pumilus (CECT5105), or left non-inoculated (controls) in two different soils, and grownundercontrolled conditions. Soil A showed a thick texture, slightly acidic with ahigh mineral nitrogen content, while soil B showed a thin texture, basic andwith a lower nitrogen content. At each sampling time, over an 8-week period,shoot and root systems of the plants were measured, nodules counted, and shootand root length and surface were determined. In addition, changes in themicrobial rhizosphere structure were evaluated by the phospholipid fatty acid(PLFA) profile extracted directly from the rhizosphere soil. The increasesdetected in shoot surface were significant only in soil A, while the rootsystemwas affected in both soils. In soil A, inoculation with B.pumilus caused a perturbation that subsequently disappeared, whilethe rhizosphere community structure was seriously altered in soil B. Allbiometric parameters were enhanced to a greater extent in soil A, in which theinoculum did not alter the existing rhizosphere communities and nutrientavailability was better.     

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.     

Field survival of containerized red and jack pine seedlings inoculated with mycelial slurries of ectomycorrhizal fungi

Red pine and jack pine seedlings growing in styroblocks were inoculated 8 wk after sowing with mycelium/agar slurries of 3 mycorrhizal fungi (Laccaria bicolor, Scleroderma citrinum, and an unidentified basidiomycete), and one suspected mycorrhizal fungus (Cantharellula umbonata). Seedlings inoculated with L. bicolor developed mycorrhizae earlier and in greater numbers than the other inoculation treatments, with red pine out-performing jack pine in both respects. At 34 wk following sowing, seedlings were outplanted on a cleared xeric site in Baraga Co., in Michigan's Upper Peninsula. Seedlings inoculated with C. umbonata failed to form mycorrhizae and were not outplanted. Inoculation treatments did not affect shoot or root weight at outplanting. Red pine inoculated with L. bicolor averaged 21% and 19% greater survival compared with control seedlings after one and two years in the field, respectively. Other inoculation treatments failed to increase seedling survival for either tree species. Jack pine demonstrated higher overall survival than did red pine for both years in all corresponding inoculation treatments.     

Growth of Nursery-grown Bamboo Inoculated with Arbuscular Mycorrhizal Fungi and Plant Growth Promoting Rhizobacteria in two Tropical Soil Typeswith and without Fertilizer Application

A nursery experiment was conducted to assess the effect of bioinoculants (Glomus aggregatum, Bacillus polymixa, Azospirillum brasilense) on seedling growth promotion of bamboo (Dendrocalamus strictus (Roxb.) Nees.) in two soil types (alfisol, vertisol) with or without fertilizer application. Bamboo seedlings were grown in the presence or absence of bioinoculants either individually or in all combinations for 180 days in field soil under tropical nursery conditions. Shoot, rhizome and root length, dry masses, nutrient concentrations and arbuscular mycorrhizal (AM) colonized root lengths were determined at harvest. Under the experimental condition tested combined inoculation of AM fungi, PSB and A. brasilense resulted in maximum growth response both under fertilized and unfertilized conditions in both soil types. Fertilizer application enhanced the efficiencies of N, P and K uptake, whereas reduced their usage efficiencies. Though soil type did not affect microbial inoculation response, fertilizer application significantly affected plant response to microbial inoculation.     

Nursery practice on seed germination and seedling growth of <Emphasis Type="Italic">Dalbergia sissoo</Emphasis> using beneficial microbial inoculants

Nursery practice using microbial inoculants was performed to find out the efficacy of the inoculants on seed germination and seedling growth of sissoo (Dalbergia sissoo Roxb.). Microbial inoculants or effective microorganisms (EM) are a mixture of many different beneficial microorganisms in a solution. The seedlings were grown in a mixture of sandy soil and cowdung (3:1) kept in polybags with pouring EM solution at different concentrations (0.1%, 0.5%, 1%, 2%, 5% and 10%) before and after a week of sowing the seeds. Seed germination rate and growth parameters of seedlings were measured, such as, shoot and root length, vigor index, fresh and dry weight of shoot and root and total biomass increment. The nodulation status influenced by EM was also observed along with the measurement of pigment contents in leaves. The highest germination rate (69%) was observed in 2% EM treatment, followed by 67% and 65% in 1% and 5% EM. The highest shoot length (33.2 cm) was in 2% EM, whereas highest root length (26.3 cm) was in 1% EM. Both fresh and dry weights from shoot and root, were maximum (4.16 g and 1.57 g; 2.12 g and 0.83 g respectively) in 2% EM and were significantly (p ≤ 0.05) different from control. Vigor index was highest (4071) in 2% EM, which was significantly (p ≤ 0.05) different from control. Total dry weight increment was highest in 2% EM treatment, followed by 1% and 5% concentrations of EM. Nodulation number was higher at very low (0.1%) concentration of EM solution but it normally decreased with the increase of EM concentrations. The contents of chlorophyll a, chlorophyll b and carotenoid were highest (60.11, 17.05 and 42.48 mg·L⁻¹ respectively) in 2% EM treatment and lowest (39.35, 13.55 and 27.29 mg·L⁻¹ respectively) in control treatment. Therefore, low concentration of EM (up to 2%) can be used for getting maximum seed germination rate and seedling development of Dalbergia sissoo Roxb.     

Effect of an ectomycorrhizal fungus,Laccaria laccata,on Fusarium damping-off in Pinus banksiana seedlings

Damping-off caused by Fusarium oxysporum Schlecht in Pinus banksiana Lamb, was reduced significantly when inoculated with an ectomycorrhizal fungus, Laccaria laccata (Scop. ex Fr.) Berk. and Br. In paired culture, growth of F. oxysporum was significantly reduced by L. laccata. The number of colony forming units of F. oxysporum was reduced significantly in the rhizosphere of P. banksiana seedlings when inoculated with L. laccata. Spore germination and germ tube length of F. oxysporum was inhibited strongly by culture filtrate of L. laccata and root exudate of P. banksiana inoculated with L. laccata. Mycorrhizal seedlings had significantly higher amount of total soluble phenols than nonmycorrhizal ones.     

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 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.