Effect of nitrogen and carbon sources on lignin and cellulose degradation by Armillaria ostoyae

Lignin has been hypothesized to be the primary mechanism of resistance to fungal pathogens in plant tissue. Degradation of lignin and cellulose by Armillaria ostoyae cultured for six weeks in Melin-Norkrans medium containing various nitrogen and carbon sources was measured radiometrically. No consistent pattern of lignin or cellulose degradation was found, regardless of A. ostoyae isolate, nitrogen source and concentration, or carbon concentration. More lignin was degraded as the concentration of glucose and fructose increased but not when the concentration of sucrose increased.     

外生菌根真菌彩色豆马勃优良菌株营养源的研究*

通过Ｐ．ｔ．优良菌株９１０９在不同培养基上生长速度、培养特性及其生长规律的研究，筛选出生长最佳培养基作为基础培养基，进行Ｃ、Ｎ营养研究，试验结果表明：Ｐ．ｔ．９１０９不仅能利用单糖、低聚糖和无机Ｎ等速效Ｃ、Ｎ源，而且能利用多糖、多元醇和有机Ｎ等长效Ｃ、Ｎ源。其菌丝生长最适Ｃ源是玉米粉和甘露糖；最适Ｎ源是蛋白胨和氯化铵。在无机Ｎ利用中，对硝态Ｎ和铵态Ｎ的利用无显著差异。应用Ｌ１６（４５）正交设计确定了Ｐ．ｔ．９１０９菌丝生长的最佳营养配方，菌丝增殖高达８６０倍，不但提高菌丝生物产量，而且节省了原料，降低了成本，为改进Ｐ．ｔ．菌剂生产配方和研制新型菌剂提供了最佳营养条件。     

桑树轮纹病病原菌的分离鉴定及其生物学特性研究

2012年6～8月,通过对桑树轮纹病病原菌分离、田间致病性测定、病原菌形态观察,将该病原菌鉴定为桑膝节霉,并对此病病原菌的生物学特性进行了研究。结果表明,不同培养基配方、温度、酸碱度、碳源、氮源、光照对桑树轮纹病病原菌菌丝的生长有一定影响,菌丝生长的最适宜培养基是理查培养基,最适温度是25℃,最适pH值是7.0,最适碳源为葡萄糖,最适氮源为蛋白胨,光照对菌丝生长无显著性差异,菌丝的致死温度和时间分别为85℃和10 min。对该病病原菌生物学特性研究为云南首次报道,对桑树轮纹病的防治具有一定的参考价值。     

The influence of carbon nutrition on Armillaria ostoyae growth and phenolic degradation

Concentrations of 1, 5 and 25 g C L^?1 as glucose, fructose and sucrose were added to basal media containing 0 or 1.0 mM catechol orwa-hydroxybenzoic acid. Media were poured in petri dishes and inoculated with one of three isolates of Armillaria ostoyae(Romagn.) Herink. Armillaria ostoyae isolates usually had greater colony size and biomass yield when glucose was the carbon source as compared to fructose or sucrose. When compared to A. ostoyae growth on basal media alone, 1.0 mM catechol and /wvi-hydroxybenzoic acia added to basal media inhibited A. ostoyae growth. In a second experiment, catechol andpara-hydroxybenzoic acid degradation by three A. ostoyae isolates growing on 1, 5 and 25 g C L^?1 glucose, fructose or sucrose were measured radiometrically. Carbon source and concentration had no effect on the degradation of pzra-hydroxybenzoic acid or catechol by Armillaria ostoyae. Results of these experiments suggests that A. ostoyae can grow faster when at high carbon concentration but cannot more effectively degrade catechol or para-hydroxybenzoic acid.     

Arbuscular mycorrhizae and ectomycorrhizae in native tree legumes in Uruguay

The level of colonization by ectomycorrhizae (ECM) and arbuscular endomycorrhizae (AM) of 23 species of native trees and shrub legumes was studied. Root samples were obtained from different regions of Uruguay. Colonization level was determined by observing AM fungal structures (hyphae, arbuscules, vesicules) in stained root segments. The number and morphological type of spores were determined in 100 g of rhizosphere soil. Ectomycorrhizal colonization was evaluated by direct and microscopic observation of root tips. All species had a high level of AM colonization. In Papilionoideae colonization varied between 62% and 78%, in the Mimosoideae between 18% and 69% and in the Caesalpinioideae the variation was larger (6–74%). These results show a significant presence of fungi that form arbuscular mycorrhizae in our soils. The number of spores forming AM in rhizosphere soil (280–l.620 spores/100 g of dry soil) was also highly relative to references for other leguminous trees. Spores were grouped in three principal morphological types Acaulospora, Glomus and Sclerocystis. Presence of ECM fungi was evident only when observed under the microscope. 26% of the plant species exhibited this type of mycorrhizae, reaching a maximal value of 36% in Mimosoideae. Only six plant species exhibited both types of mycorrhizae. Dual colonization could help in the colonization of poor soils and for their use in agroforestry systems.     

Role of metal proteinases in the fruit-body formation ofHypsizygus marmoreus

We investigated the possible role of metal proteinase on the fruit-body formation ofHypsizygus marmoreus. The addition of a specific metal proteinase inhibitor, phosphoramidon, to the culture medium (10g/ml) completely inhibited fruit-body formation. Metal proteinase activity in both the medium and the mycelia of this fungus increased markedly during vegetative mycelial growth, and activity was maximal 25 days after inoculation. When phosphoramidon was added to the culture medium during vegetative mycelial growth, the metal proteinase activity in the mycelium decreased to 56% of the control (without inhibitor) level. Isoelectric focusing analysis showed that two kinds of metal proteinases with a pl of 7.7 and 8.4, respectively, were obtained from 29-day-old mycelia. Uptake of phosphoramidon into the mycelia was confirmed as the result of inhibition of thermolysin activity by the mycelial extracts. The degree of inhibitor uptake into mycelia was about 2.0% and was independent of the initial concentration of the inhibitor administered. The addition of peptone and amino acids to medium treated with phosphoramidon resulted in fruit-body dry weight yields that were about 50% that of the control.     

Evaluation of Pinus radiata seed treatments to control Fusarium circinatum: effects on seed emergence and disease incidence

In this study, the effects of hot water (HWT), hydrogen peroxide and fungicides on the incidence of Fusarium circinatum on artificially inoculated Pinus radiata seeds were evaluated. Fifteen commercial fungicide formulations were screened in vitro for inhibitory activity on mycelial growth and conidial germination of F. circinatum. With half‐maximal effective concentration (EC₅₀) lower than 0.5 ppm, fluazinam, imazalil and tebuconazole were the most effective fungicides on mycelial growth, while captan, mancozeb or pyraclostrobin were the most effective (EC₅₀ < 0.3 ppm) on conidial germination. Based on the results obtained, imazalil, fluazinam, mancozeb and pyraclostrobin were selected for further testing. The effects of HWT, hydrogen peroxide and fungicide treatments on seed emergence and the incidence of F. circinatum were assessed. Seed treatments with fungicides prior to sowing were less effective and inconsistent in reducing the incidence of F. circinatum on seedlings. In contrast, hot water and hydrogen peroxide treatments significantly reduced F. circinatum contamination on P. radiata seeds with an overall disease incidence lower than 0.8% on seedlings. Furthermore, subsequent application of fungicides on seedlings did not improve the effectiveness of HWT. These results, therefore, suggest that hot water is a better alternative to hydrogen peroxide and fungicides as Pinus seed treatment against F. circinatum and could easily be implemented as standard in commercial nurseries to control the spread of the pitch canker disease.     

落叶松外生菌根真菌优良菌株筛选及其在造林中的应用

落叶松外生菌根真菌优良菌株筛选及其在造林中的应用周玉芝，韩桂云，齐玉臣，刘忱，何兴元关键词菌根真菌，落叶松，造林菌根真菌与树木共生营养，对提高育苗、造林成活率及林木速生具有重要的经济意义。为了研究东北地区主要造林树种─—落叶松的菌根真菌及选育优良菌株...     

Antifungal properties of terpenoids in Picea abies against Heterobasidion parviporum

The antifungal properties of 14 major oleoresin‐constitutive terpenoids of Norway spruce (Picea abies) against Heterobasidion parviporum were evaluated in vitro at three gradient concentrations, 0.1, 0.2 and 0.4 μmol/cm², on potato dextrose agar medium. Eight monoterpene hydrocarbons (+)‐ and (?)‐α‐pinene, (?)‐β‐pinene, (+)‐3‐carene, myrcene, (+)‐ and (?)‐limonene, terpinolene; four oxygenated monoterpenes α‐terpineol, terpinen‐4‐ol, 1,8‐cineole, bornyl acetate; and two resin acids abietic acid and dehydroabietic acid were selected. Abietic and dehydroabietic acids showed the highest antifungal activities at all concentrations. Among oxygenated monoterpenes, bornyl acetate and α‐terpineol showed antifungal activity at the highest concentration. Among monoterpene hydrocarbons, (+)‐α‐pinene showed similar activity to terpinen‐4‐ol and 1,8‐cineole at the highest concentration; however, it was lower than α‐terpineol and bornyl acetate. Other monoterpene hydrocarbons inhibited mycelial growth by <10%. Re‐extraction of medium surfaces after the test period revealed that most α‐terpineol and terpinen‐4‐ol remained unchanged on the surface but monoterpene hydrocarbons completely disappeared. However, notable fungal transformed products were observed on surfaces applied with 1,8‐cineole and bornyl acetate. Thus, mycelial growth inhibition of monoterpenoids might be caused by complex functions such as biodegradation and/or detoxification by interaction between mycelium and compound. These results provide a basis for future studies considering the role of chemodiversity in the comprehensive chemical defence of P. abies.     

Bacterial strains isolated from cedar wood improve the mycelial growth and morphology of white rot fungus <Emphasis Type="Italic">Phlebia brevispora</Emphasis> on agar and liquid medium

Growth enhancement of white rot fungus Phlebia brevispora TN3F by two bacterial strains TN3W-8 and TN3W-14 which showed high identity with genus Pseudomonas and Enterobacter, respectively, was investigated in liquid and solid media at different glucose concentrations. The white rot fungus P. brevispora TN3F and the bacterial strains were isolated from same white rot-decayed cedar wood from the Tano Forest Science Station at University of Miyazaki. Confrontational assay between the fungus and the bacterial strains revealed a fungal growth enhancement phenomenon, in the direction of the bacterial cells, even without direct contact. This enhancement increased with decrease of glucose concentration, 0.5% glucose offering comparatively the most mycelial growth. In potato extract (PE) medium at 0.5% glucose concentration, along with mycelial growth enhancement and morphological change of P. brevispora TN3F, growth and morphological enhancement of fungus TMIC33929, another strain of P. brevispora, was achieved by the bacterial strain TN3W-14, although TMIC33929 originated from a different environment. Outside few reports of a positive interaction between white rot fungi and other microorganisms, this is a first report of such positive interaction in liquid medium. Furthermore, this also is the first report of a single bacterium having potential to positively affect mycelia growth of different strains of a white rot fungus.     

Macrofungal diversity of holm-oak forests at the northern limit of their distribution range in the Iberian Peninsula

Given the current gap in knowledge concerning the macrofungal diversity of holm-oak forests at the northern limit of their distribution range and in the Basque Country, a mycocoenological study was conducted by sampling 23 randomly selected permanent plots from 6 holm-oak forests. To obtain a better understanding of the macrofungal diversity distribution pattern on a regional scale, forests from both the temperate and Mediterranean-type climate regions of the Basque Country were selected, covering all their phytocoenological variation. After 4 years of sampling, a total of 377 macrofungal species were collected: 209 ectomycorrhizal species, 86 humicolous saprotrophs, 40 saprotrophs on litter, 36 lignicolous saprotrophs, 3 coprophilous saprotrophs and 3 parasitic species. Species considered characteristic of this kind of forest, such as Cortinarius aurilicis, Cortinarius callochrous, Cortinarius ionochlorus, Hygrophorus leucophaeo-ilicis, Hygrophorus persoonii, Hygrophorus russula, Lactarius atlanticus, Lactarius ilicis, Leccinellum lepidum, were recorded all over the territory, but Mediterranean forests were richer in species and more productive in sporocarps than temperate ones. Species assemblage was found to change gradually at local scale from temperate to Mediterranean climate zones, and was distinct to each phytocoenological association with all species, with ectomycorrhizal species and with saprotrophs.     

Analysis of Norway spruce dieback phenomenon in Latvia – a belowground perspective

In 2010, dieback of Norway spruce (Picea abies) was observed in Latvia. As mostly stands on seasonally or permanently water-saturated soils were damaged, we hypothesized that the recorded foliar damage might be associated with belowground factors. We established 48 sample plots in stands with damage symptoms for soil parameter evaluation and fine root sampling to determine the number of fine roots in different growth stages and to describe the associated fungal community. Among sample plots on organic soils, there were fewer viable older fine roots and greater number of dead fine roots in more damaged sites than in less damaged. These root parameters were significantly correlated with higher groundwater level and soil pH. The fungal community was dominated by ectomycorrhizal (ECM) species (the most common was Tylospora asterophora), the endophyte Oidiodendron maius and saprotrophic species Cryptococcus magnus. Saprotrophic species had higher abundance in more damaged sites, suggesting a shift in fungal communities from ECM fungi. In conclusion, the results of this work suggest Norway spruce root system response to short-term climatic stress, which should be considered when planning spruce forest management, especially on organic soils with a high groundwater level.     

木麻黄根瘤中分离的三株弗兰克氏菌生长与培养方法的研究

比较了从不同来源的木麻黄根瘤中分离得到的三株弗兰克氏菌在两种浓度不同碳源培养基中的生长差异，结果表明：供试菌在以丙酮酸钠为唯一碳源时生长最好，其生长量为其它碳源的１．３－４７．７倍；最适合的碳源浓度为０．２３ｇＣ＼Ｌ。在三种不同的Ｆｒａｎｋｉａｘ菌地，通入无菌空气发酵培养培养能显著地提高弗兰克氏菌的生长量。同时还探讨了弗兰克氏菌的发酵条件。     

Phenotypic characterization of cryptic Diplodia species by MALDI‐TOF MS and the bias of mycelium age

Detection and identification of fungal cryptic species has been facilitated by DNA sequencing. However, the examination of some phenotypic traits is fundamental for the confirmation of genetic results. MALDI‐TOF mass spectrometry has shown remarkable reliability in the recognition of species‐specific phenotypic markers and has already been tested in several species of fungi. However, even though there is no direct evidence, the age of the mycelium appears to influence the composition of the metabolites detected. In this study, we demonstrate that MALDI‐TOF is a reliable technique to identify suitable metabolites to distinguish three monophyletic species of Diplodia (D. pinea, D. seriata and D. scrobiculata), thus supporting recent DNA results. Nevertheless, different collections of samples over a period of 3 months after inoculation also revealed that the MALDI‐TOF spectra are highly dependent on mycelial age. More importantly, the species‐specific markers emerge only after 1 month of mycelial growth. The methodological and biological implications of these findings are discussed.     

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.     

添加葡萄糖对杉木人工林土壤氮素转化及净矿化和硝化的影响

以亚热带杉木人工林为研究对象,研究添加葡萄糖(C量水平分别是0,100,300,1 000,2 000,5 000 mg·kg-1)对土壤氮含量、氮素矿化和硝化的影响。结果表明,葡萄糖添加降低土壤无机氮含量和比例,硝态氮的降低幅度大于铵态氮;但是没有降低可溶性有机氮(SON)和pH值,甚至提高SON的比例。添加葡萄糖降低氮素净矿化和硝化速率,氮素矿化作用受到抑制。结果显示,随着葡萄糖添加,亲水性氮所占比例显著降低,这与氮的固持和转化有关,导致SON比例增加;分析表明,硝态氮和可溶性有机氮在提取液全氮中所占比例成显著的线性负相关关系(R2=0.902)。研究发现,1 000 mg·kg-1的葡萄糖C添加量可能是影响杉木人工林土壤氮素转化的分界点。     

Wood-inhabiting fungi in stems of Fraxinus excelsior in declining ash stands of northern Lithuania,with particular reference to Armillaria cepistipes

Abstract

Stem bases of 210 Fraxinus excelsior trees of three different health categories were sampled by the means of an increment borer in declining ash stands in northern Lithuania. From this number, 15 sound-looking, 132 declining and 63 dead trees from three discrete plots yielded 352 isolates, representing 75 fungal species. In addition, mycelial fans and rhizomorphs typical of Armillaria spp. from 205 and 20 trees, respectively, were sampled and subjected to fungal isolations. Species richness was similar in trees from each health category, but community structures differed, indicating that species composition of wood-inhabiting fungi in stems changes along with the changes in tree health condition. Armillaria cepistipes was the most common species (86 isolates from 210 wood samples, or 41.0%), isolated more frequently and consistently than any other potential tree pathogen. It also showed abundant occurrence on a majority of trees in the form of mycelial fans and rhizomorphs, from which 64 and 14 isolates of the fungus were obtained, respectively. The population structure of A. cepistipes revealed the presence of 53–93 genets per hectare, some of which extended up to 30–55?m.     

Chemical control of two Phytophthora species infecting the canopy of Monterey pine (Pinus radiata)

Twelve active ingredients were screened for their ability to control foliage disease caused by Phytophthora kernoviae and Phytophthora pluvialis. Inhibition of mycelial growth and sporangial production of both pathogens were assessed in in vitro assays after exposure to three concentrations of each active ingredient. While most of these active ingredients inhibited mycelial growth in vitro, due to their widespread use, phosphite, copper oxychloride and metalaxyl‐M were selected for further study. Four rates of each active ingredient were applied to two Pinus radiata genotypes, and detached needle assays at 6 and 90 days post‐treatment were used to determine treatment efficacy and persistence. Untreated needles showed significantly larger lesions than all fungicide‐treated needles after exposure to P. pluvialis or P. kernoviae on both sampling dates. Efficacy and persistence of the three active ingredients did not increase with increase in concentration. Larger lesions were formed on more susceptible genotype after inoculation with both Phytophthora species, even when higher rates of active ingredients were applied. Phosphite, copper oxychloride and metalaxyl‐M have potential to protect commercially planted P. radiata from these two Phytophthora species.     

The importance of wild plant species as potential inoculum reservoirs of white root rot disease

Rosellinia necatrix is a soilborne plant pathogenic fungus belonging to the phylum Ascomycota. The fungus is distributed widely in temperate zones and often causes huge economic losses on numerous crop plants. To determine the potential for wild plants to serve as inoculum reservoirs of white root rot disease, eight woody and two herbaceous plant species belonging to nine families were inoculated with two R. necatrix isolates. The species used were Actinidia polygama, Broussonetia kazinoki, Camellia sinensis, Castanea crenata, Hydrangea serrata, Magnolia obovata, Pteridium aquilinum, Petasites japonicus, Quercus serrata and Rosa multiflora, all common flora in the cool temperate forests of Japan. The mortality of plants varied between species, independent of the effect of fungal isolates. Although some of the plants survived fungal infection, all plant species tested were more or less susceptible, having rotting lesions on the inner bark and cortex. For the woody plants, most of the mycelial strands in the diseased tissues became plumose mycelia, growing compressed between the outer and inner barks. However, mycelia found in diseased tissues of the two herbaceous plants were not in the form of plumose mycelia. All plant species harboured mycelial strands on the surface of their root systems and underground main stem, and the amount of the mycelial strands did not correlate with the mortality of the plants. Even if plants exhibited no external symptoms, some of them harboured abundant mycelial strands. In conclusion, wild plants can serve as alternative host plants for R. necatrix and function as persistent inoculum reservoirs, irrespective of their apparent health status. Thus, assessments of such latent host plants must be considered in the course of managing white root rot.