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.     

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.     

No “Gadgil effect”: Temperate tree roots and soil lithology are effective predictors of wood decomposition

The “Gadgil effect” hypothesizes that root associations may slow down decomposition through pre‐emptive competition. In the context of recalcitrant litter decomposition, specifically coarse wood debris, it is uncertain as to what is the relative importance of soil communities associated with living roots when compared to those without roots. Here, it is hypothesized that the presence of live roots and active photosynthates will enhance wood decomposition. To test this hypothesis, the presence or absence of temperate tree roots was used in this study. Sugar maple (Acer saccharum) and white oak (Quercus alba) roots were manipulated at three sites of either limestone or shale parent rock residuum. At each site, wood substrate was placed in soils beneath the canopy of either A. saccharum or Q. alba, while in the presence of roots (root⁺). At the same time, wood substrate was placed in the same soil community, but live root exposure was eliminated by trenching (root^?). This eliminated active photosynthate supply to the soil microbial community. Results determined that live root exposure promoted faster decomposition and greater mycelial colonization of wood substrate. Also, sites of shale parent rock residuum had higher rates of decomposition in comparison with limestone parent rock residuum. Although additional work is needed to determine the extent in which roots and lithology can facilitate wood decomposition, these findings suggest that living roots impact decomposers and provide a pathway towards humus and soil organic matter formation.     

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.     

Effects of ectomycorrhizal fungi on damping‐off and induction of pathogenesis‐related proteins in Pinus tabulaeformis seedlings inoculated with Amanita vaginata

The formation of mycorrhiza on Pinus tabulaeformis (Pt) seedlings by ectomycorrhizal fungi (ECMF), the protective effect of the ECMF on damping‐off caused by Rhizoctonia solani and the expression of pathogenesis‐related proteins (PR‐proteins) induced by Amanita vaginata and/or R. solani in Pt seedlings were investigated. The results indicated that: (i) among the 10 ECMF tested, five species (Gomphidius viscidus, Suillus laricinus, Suillus tomentosus, A. vaginata and Basidiomycetes no. 058) formed mycorrhiza with Pt seedlings. There was a positive correlation between mycorrhizal colonization levels and protective effects (r = 0.9207). Among the five species, A. vaginata had the highest colonization level and the best protective effect against damping‐off; however, G. viscidus had no protective effect even though it had a high mycorrhization level. (ii) Chitinases, β‐1,3‐glucanases and PR‐proteins in the seedlings were not induced by A. vaginata colonization, but were induced when A. vaginata‐mycorrhizal seedlings were attacked by R. solani. It is possible that chitinases, β‐1,3‐glucanases and 108, 97, 78, 44, 37 and 14‐kDa PR‐proteins induced both by A. vaginata and R. solani protect Pt seedlings from R. solani infection.     

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.     

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.     

Decomposition and nutrient release from leaves,twigs and roots of three alley-cropped tree legumes in central Togo

The decomposition of leaves, twigs and roots of two diameter classes (<1.5 mm, 1.5–5 mm) were examined in an alley cropping experiment withGliricidia sepium, Calliandra calothyrsus andSenna sianea in the subhumid savanna of Central Togo using the litterbag technique. The effect of the application of leaves and twigs as mulch or green manure was examined. Gliricidia showed the most rapid mass loss. For all species, leaves decomposed faster than roots. Twigs had the lowest decomposition rate except forCalliandra. The two diameter classes of roots decomposed differently in the three species:Gliricidia fine roots decomposed faster than its coarser root fraction, the coarse roots ofSenna decomposed faster than the fine roots.Termites influenced the mass loss of twigs and roots to varying extents for the different species. In the case of twigs this was markedly influenced by the mode of application: green manure showed more termite frass than mulch.Nutrient release resembled the mass loss patterns of the prunings except for K, which was leached independently from mass loss. The release of the different nutrients was in the order CaGliricidia andSenna seemed to be best for mulch and green manure production at our site.     

The effect of nitrogen fertilization on fungistatic phenolic compounds in roots of beech (Fagus sylvatica) and Norway spruce (Picea abies)

The effect of nitrogen fertilization on fungistatic phenolic compounds in fine roots of beech and Norway spruce growing in afforestation plots was analysed. The plots were situated at two sites in Switzerland on acidic soil with low base saturation. For 9 years, the trees have been treated with dry ammonium nitrate to give 0, 10, 20, 40, 80, 160 kg N ha^?1 year^?1, respectively. The phenolic compounds responded differently to fertilization. Fine roots of beech showed a significant decrease of (?)‐epicatechin and piceatannol with increasing nitrogen fertilization. The concentration of protocatechuic acid was increased with fertilization. Roots of fertilized Norway spruce showed significantly decreased concentrations of 4‐hydroxyacetophenone and piceatannol. The mycelial growth of three isolates each of Heterobasidion annosum s.l. and Cylindrocarpon destructans was tested on agar media containing various phenolic compounds in concentrations found in fine roots of Norway spruce (Picea abies) and beech (Fagus sylvatica). All three H. annosum isolates were inhibited by p‐coumaric acid and (?)‐epicatechin. Two isolates were inhibited by another four phenolic compounds (p‐hydroxybenzoic acid, 4‐hydroxyacetophenone, piceatannol and protocatechuic acid), one by (+)‐catechin. Two of three C. destructans isolates were inhibited by all phenolic compounds except for (+)‐catechin which affected only one isolate, one isolate did not respond at all.     

Root trenching: a useful tool to estimate autotrophic soil respiration? A case study in an Austrian mountain forest

We conducted a trenching experiment in a mountain forest in order to assess the contribution of the autotrophic respiration to total soil respiration and evaluate trenching as a technique to achieve it. We hypothesised that the trenching experiment would alter both microbial biomass and microbial community structure and that fine roots (less than 2 mm diameter) would be decomposed within one growing season. Soil CO₂ efflux was measured roughly biweekly over two growing seasons. Root presence and morphology parameters, as well as the soil microbial community were measured prior to trenching, 5 and 15 months after trenching. The trenched plots emitted about 20 and 30% less CO₂ than the control plots in the first and second growing season, respectively. Roots died in trenched plots, but root decay was slow. After 5 and 15 months, fine root biomass was decreased by 9% (not statistically different) and 30%, (statistically different) respectively. When we corrected for the additional trenched-plot CO₂ efflux due to fine root decomposition, the autotrophic soil respiration rose to ~26% of the total soil respiration for the first growing season, and to ~44% for the second growing season. Soil microbial biomass and community structure was not altered by the end of the second growing season. We conclude that trenching can give accurate estimates of the autotrophic and heterotrophic components of soil respiration, if methodological side effects are accounted for, only.     

Recovery of a phytopathogenic bacterium Lonsdalea quercina from a lesser horseshoe bat in Moravian karst,Czech Republic

While bats are important reservoirs and vectors of pathogenic agents with zoonotic potential, their skin‐associated microbial community could harbour organisms relevant to bat health as well as those associated with bat habitats in general. A total of 140 skin swabs collected from bats in the Czech Republic were cultured for bacteria from 2012 to 2013. A phytopathogen, isolated from a lesser horseshoe bat (Rhinolophus hipposideros), was identified as Lonsdalea quercina by MALDI‐TOF (matrix‐assisted laser desorption–ionization time‐of‐flight mass spectrometry) and characterized as Lonsdalea quercina subsp. britannica using phylogenetic analysis based on a 16S rRNA and two housekeeping (infB and atpD) genes. Lonsdalea quercina subsp. britannica is a gram‐negative facultative anaerobic bacterium reported to cause bark canker and drippy nut disease in oaks. Our finding provides evidence of Lonsdalea quercina in the Czech Republic (Moravian Karst). As the lesser horseshoe bat is a sedentary species, it may bio‐indicate the presence of the phytopathogenic bacterium in the broadleaved forests of the habitat of pathogen recovery. We therefore suggest active surveillance of the area of the Moravian Karst for signs of oak disease and/or decline.     

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.     

A method for observing and manipulating roots and root associated fungi on plants growing in nonsterile substrates

Standard 14‐cm plastic‐petri dishes were used as root substrate chambers allowing controlled inoculation, regular inspection under the dissecting microscope, and manipulation of root infections with pathogens and mycorrhizal fungi under nonsterile conditions. Roots of young pine seedlings were placed on the root substrate and, especially those growing on the substrate surface under the lid, were very easy to manipulate. Mycorrhizal species and inoculation methods were compared and the infection process followed continuously under various conditions. The air pockets formed between the lid and the substrate favoured mycorrhiza formation. Brief, periodic flooding prevented formation of mycorrhizas in two mycorrhizal species having hydrophobic hyphae but did not prevent it in less hydrophobic species. IAA treatment of roots led to heavy production of mycorrhizas. Fruiting body formation in Lacearia laccata was induced and followed in the dish cultures. Pure mycorrhizal stock cultures were able to be revitalized in the dish cultures and reisolated in pure culture. The infection strategy of a weak root pathogen, Cylindrocarpon destructans, was studied with the same technique, which was also used for investigating treatments predisposing seedlings to pathogen infection.     

Involvement of Phytophthora species in white oak (Quercus alba) decline in southern Ohio

This study was initiated to investigate the possible role of Phytophthora species in white oak decline (Quercus alba) in southern Ohio at Scioto Trail State Forest. Surveys demonstrated the presence of four species of Phytophthora including one novel species. By far, the most common species was P. cinnamomi; P. citricola and P. cambivora were isolated infrequently. In few instances, P. cinnamomi was isolated from fine roots and necroses on larger roots. No special pattern of incidence was found, but P. cinnamomi was more commonly isolated from greater Integrated Moisture Index values suggesting moist lower bottomlands favour this Phytophthora species. When tree crown condition was examined relative to the presence of Phytophthora, no significant association was found. However, roots of declining P. cinnamomi‐infested trees had 2.5 times less fine roots than non‐infested and healthy trees, which was significantly different. The population densities of P. cinnamomi from declining trees were significantly greater than from healthy trees, suggesting increased pathogen activity that has the potential to cause dieback and decline and possibly the cause of a reduced fine root amount found on declining trees.     

Influence of site factors on the impact of Phytophthora cinnamomi in cork oak stands in Portugal

Although decline of cork (Quercus suber) and holm oak trees (Quercus rotundifolia) has been described in Portugal in the late years of the 19th century, its development has become a motive of high concern during the last two decades. The presence of Phytophthora cinnamomi in cork and holm oak stands was surveyed in four different regions of the country (Trás‐os‐Montes, Alentejo, Ribatejo and Algarve) during 1995–98. Tree decline severity, sudden death and site characteristics were assessed in 56 sites representing varied conditions. The pathogen was isolated from oak roots and rhizosphere samples in 27 of those places. Other plant species from natural vegetation were sampled in three active disease centres. This survey showed that 56% of the surveyed species of shrub flora were infected with P. cinnamomi, which was detected mainly on species belonging to the families Ericaceae, Cistaceae and Leguminosae. Recovery of P. cinnamomi was more frequent in shallow soils (Leptosols and complex Leptosols and Luvisols). These soils are more common in the south (Algarve), where decline has a high impact. Soils with low fertility and low mineral nutrient levels, particularly phosphorus, seemed to favour infection. Site aspect and topographic tree situation were also evaluated. Sites facing south showed higher occurrence of P. cinnamomi, which was also more frequent in slopes and valleys than on hilltops. In Algarve, a relationship could be established between the crown status and the presence of P. cinnamomi in roots and rhizosphere. Different morphotypes of P. cinnamomi could be distinguished in vitro, and their occurrence in the field was correlated with particular site characteristics. Further research needs and management strategies to limit the extension of the disease are discussed.     

Rooting and early shoot characteristics of Picea abies (L.) karst. cuttings originating from shoots with enforced vertical growth

Picea abies mother‐plants from 10 serially propagated clones (14 years old, chronologically) were treated in four different ways: (1) control with intact, non‐manipulated plants; (2) like (1) but with the leader cut off; (3) intact plants but with all branches of the upper two whorls forced to grow in a vertical way; and (4) like (3) but with the leader cut off. Cuttings from four positions (cutting‐types) on the mother‐plants were collected, rooted and evaluated for rooting %, number of main roots, number of lateral roots, plagiotrophy, needle symmetry on leader and growth of leader. Treatments 3 and 4 resulted in cuttings with more plagiotropic growth but with better rooting, more lateral roots and better growth than did cuttings from treatments 1 and 2. The genotype had a significant influence on rooting. Number of main roots was significantly influenced by the genotype and the cutting‐type, but not of the mother‐plant treatment. Symmetry of needles on the leader was fairly weakly influenced by genotype and cutting‐type yet not influenced by mother‐plant treatment. Clone effect was significant for leader growth.     

Studies on root distribution of five multipurpose tree species in Doon Valley,India

Observations on the growth performance, rooting behaviour and distribution of fine roots of five tree species viz., Bauhinia purpurea, Grewia optiva, Eucalyptus tereticornis, Leucaena leucocephala and Ougeinia oojeinensis (Family: Papilionaceae) are being presented here. Roots were exposed at the time of planting, 6 months, 16 months, and 28 months after planting. Total root weight and root volume were highest in Eucalyptus tereticornis and lowest in Bauhinia purpurea. Major part of the root system confined within 90–120 cm soil depth in case of Bauhinia purpurea, Grewia optiva and Leucaena leucocephala but Eucalyptus tereticornis and Ougeinia oojeinensis strike their roots to deeper depths. Bauhinia purpurea had its roots evenly distributed down to 120 cm. In general, the vertical distribution of fine roots (< 2 mm in diameter) of the five species indicate that more the depth, fewer the number of roots. The observations on soil binding capacity, indicated that Ougeinia oojeinensis had the maximum and Eucalyptus tereticornis had the minimum binding value. Due to leaf shading and other litter fall significant increase of nutrient components in soils under the tree canopies has been observed. The study indicates that bulk of the roots of the five tree species are found near the surface, but observation on soil moisture and nutrient content does not indicate variation under the tree canopies and in open, hence there may not be root competition in initial years of plantation.Authorised for publication by the Institute as contribution No.3311/239/89.     

Pathogenicity and virulence of Armillaria sinapina and host response to infection in Douglas‐fir,western hemlock and western redcedar in the southern Interior of British Columbia

The frequency of infection, lesion characteristics and anatomical changes in phloem and cambial tissues caused by Armillaria sinapina were studied on inoculated trees of Douglas‐fir, western hemlock and western redcedar and compared with results of A. ostoyae inoculations previously reported on the same host species. Similar percentages of inoculations resulting in infection of roots on the three hosts indicate that A. sinapina and A. ostoyae are equally pathogenic. Armillaria ostoyae was more virulent than A. sinapina as demonstrated by fungal exudates from A. ostoyae inoculum blocks, which appeared to cause lesions on roots; the higher frequency at which lignified impervious tissue (IT) and necrophylactic periderm (NP) developed in bark and following cambial invasion, compartmentalization; the large proportion of roots that showed no visible host response; the large zones of IT formed under continuous stimulation by A. ostoyae advancing in inner bark; and the high frequency of breaching of NP barriers. Spread of A. sinapina mycelium in host species appeared slower than that of A. ostoyae, particularly in Douglas‐fir and western hemlock. In western redcedar, A. ostoyae induced stronger host responses than those following invasion by A. sinapina, which included further expansion of the induced rhytidome response, traumatic phloem resin duct formation and higher numbers of polyphenolic parenchyma comprising its barrier zone. Where damage by A. sinapina ensued, it was always associated with high inoculum potential. The ecology of virulent and less virulent species of Armillaria in natural forests is discussed.     

Necrophylactic periderm formation in the roots of western larch and Douglas‐fir trees infected with Armillaria ostoyae. II. The response to the pathogen

Periderm formation was studied in bark samples collected from the roots of western larch (Larix occidentalis) and Douglas‐fir (Pseudotsuga menziesii) trees infected with Armillaria ostoyae. Necrophylactic periderms were formed in advance of infection and successfully restricted continued fungal spread in 68 and 45% of the samples collected from 10‐ and 27‐year‐old western larch, respectively. However, all periderms formed in 11‐ and 25‐year‐old Douglas‐fir had been breached by the advancing fungus. In both species, necrophylactic periderms were commonly breached at the junction of the periderm with the vascular cambium. In western larch, stone phellem often comprised the external phellem layer of necrophylactic periderms with multiple bands of phellem. In 27‐year‐old western larch, infection was often confined to discrete lesions bounded by multiple periderms with multiple bands of phellem. In both tree species, phellem production was greater in response to infection than in response to abiotic wounding.     

Characteristics of soil microbial biomass and community composition in Pinus yunnanensis var. Tenuifolia secondary forests

Pinus yunnanensis var. Tenuifolia is an important species of timber and grease in southern China, but the characteristics of the soil microbial community in P. yunnanensis var. natural secondary forests are still poorly understood. Using a fumigation-extraction method and phospholipid fatty acid (PLFA) analysis, we study microbial biomass and community composition in the topsoil (0–10 cm) of three types of secondary forests (PYI, PYII, PYIII) dominated by P. yunnanensis var. to varing degrees. Microbial biomass carbon and nitrogen, total PLFA, and PLFA contents of bacterial, fungal, and arbuscular mycorrhizal fungi were significantly lower in PYI than PYII or PYIII, and there were significant differences in the monounsaturated/saturated fatty acid ratio among the tested forests. Principal component analysis indicated that the soil microbial community structure of the tested forests differed significantly. The changes in soil microbial biomass and community composition were positively correlated with soil water content, pH, organic matter (SOM), total nitrogen (TN), and total phosphorus. Season did not significantly affect the soil microbial community structure, but significantly affected soil microbial biomass, SOM, and TN, which were higher in the dry season than in the wet season.