The comparison of isolation techniques for nematophagous fungi from soil

Using a single soil, a comparison was made of six well-known techniques for the isolation of nematophagous fungi. Each technique was tested for its ability to isolate the fungi present with regard to the number of species recovered, the number of plates needed to be 95% sure of isolating all the species possible and the amount of soil required. The advantages and disadvantages of each method are discussed. The results show that specific techniques are required for the isolation of endoparasites and predators respectively. The soil sprinkling technique and the Baermann funnel technique are shown to be the most efficient methods overall, with six and seven replicates required for 95% probability of isolating all the predator and endoparasite species respectively.     

Effects of Storage Temperature and Extraction Procedure on Recovery of Plant-parasitic Nematodes from Field Soils

Storage of nematodes in soil at -15 C for 1 to 16 weeks greatly increased nematode recovery by a sugar-flotation-sieving procedure. One week of exposure to -15 C killed all nematodes except Pratylenchus zeae and Tylenchorhynchus claytoni which were recoverable in decreasing numbers up to 10 weeks by the Baermann funnel method. Optimum storage temperature for survival of most nematode species was 13 C. The numbers of Meloidogyne incognita, T. claytoni, Belonolaimus Iongicaudatus, and P. zeae recoverable by either extraction method remained constant or increased when stored at 13-24 C for 16 weeks. This was also true for Helicotylenchtts dihystera and Xiphinema americanum extracted by the Baermann funnel technique, whereas the numbers retrieved by the sugar-flotation-sieving method decreased slightly. All species except T. claytoni decreased appreciably in soil stored at 36 C.     

华东地区根结线虫寄生真菌调查*

通过对5种分离根结线虫寄生真菌的方法的比较,发现通过雌成虫平板分离法和卵平板分离法有较高的成功率,其他三种自幼虫上或土壤中分离的方法很少取得成功。从华东地区104个根结线虫样品中分离得到根结线虫寄生真菌638个菌株, 鉴定为14个属22个种,其中Cylindrocarpon destructans, C.heteronema, Fusarium equiseti, F.lateritium, F.proliferatum, Gliocladium virens, Humicola fuscoatra, Idriella lunata, Trichoderma hamatum, T.harzianum, Verticillium catenulatum, Volutella ciliata 为首次报道在根结线虫上寄生。通过种群分析发现Fusarium solani出现频率最高, F. oxysporum, Paceilomyces lilacinus 和 Acremonium strictum为常见种,这些种在根结线虫上广泛分布。同时还发现卵和成虫寄生真菌有所不同,Gliocladium virens 和Trichoderma harzianum仅在卵上分离到,而Volutella ciliata, Trichoderma hamatum 和Idriella lunata仅在雌成虫分离到,从幼虫上仅分离一次Paecilomyces lilacinus。其它真菌在卵和成虫上均可分离得到,真菌种类在三种根结线虫上和在不同地区有所不同。     

Extraction of nematodes from Dry Valley Antarctic soils

Nematode density and taxonomic composition from Dry Valley soil processed by the sugar centrifugation (SC) method in Antarctica was compared to those extracted from soils shipped frozen to the USA and processed by either the SC or Baermann Funnel (BF) (at 5°C and 10°C) techniques. Soil selected for the extraction comparisons represented a wide range of soil properties found in the Dry Valleys. More nematodes were recovered from freshly collected Antarctic soil and from stored frozen soil using the SC technique than from BF at either temperature (P<0.05). Temperature had no effect on nematode densities extracted by the BF. Scottnema lindsayae was the most abundant species recovered by all extraction methods, but recovery was significantly lower from stored soils. Thus, nematodes can be extracted qualitatively following frozen storage using SC, but quantitative studies of nematode populations should be based on soils extracted following field sampling.     

Soil-inhabiting Tardigrade Communities in Forests of Central Japan

This study was carried out for the purpose of detecting the relationship between soil-inhabiting tardigrade communities and environmental factors of various forests. Nine forests in the southern part of Kanagawa Prefecture, Central Japan, were selected for this study. Four vegetation types were designated; broadleaved (evergreen/deciduous), coniferous and orchard. In these sites, dry weight of leaf litter, soil pH, soil hardness, soil moisture content and porosity ratio were measured. Wet soil faunal frequencies were also described. The Baermann funnel method was adopted for collecting tardigrades, and DIC microscopy was used for specific identification. To clarify the correlation between environmental variables and tardigrade faunae, multivariate analysis was applied. The tardigrade fauna occurred as two distinct groups. The first group primarily contained Macrobiotus species. The second group contained the genus Diphascon (e.g. D. nobilei, D. patanei, D. prorsirostre). An exception to this was D. pingue, included in the former. Most of the Diphascon species were concentrated in Nebu (coniferous forest site), while, Macrobiotus species were dominant in other sites. Distinct environmental factors could not be identified, but the nematodal frequency was recognized as the main factor in forming these groups. The uniqueness of Nebu, which does not correspond to large-scaled vegetational classification, was substantiated by statistical data. Nebu’s coniferous forest apparently has created a unique environment for sustaining rare species (Diphascon species). This study concluded that forests should be evaluated not only by the macroscopic factors, such as landscape, but also by the microscopic communities, such as those including the tardigrades.     

Predicting Damage of Meloidogyne incognita on Watermelon

Quantitative growth response of watermelon (Citrullus lanatus) sensitive to Meloidogyne incognita is poorly understood. Determination of soil population densities of second-stage juveniles (J2) of M. incognita with Baermann funnel extraction often is inaccurate at low soil temperatures. In greenhouse experiments, three sandy soils were inoculated with dilution series of population densities of eggs or J2 of M. incognita and planted in small containers to watermelon ‘Royal Sweet’ or subjected to Baermann funnel extraction. After five weeks of incubation in the greenhouse bioassay plants in egg-inoculated soils, gall numbers on watermelon roots related more closely to inoculated population densities than J2 counts after Baermann funnel extraction. In April 2004, perpendicularly-inserted tubes (45-cm diameter, 55-cm deep) served as microplots where two methyl bromide-fumigated sandy soils were inoculated with egg suspensions of M. incognita at 0, 100, 1,000 or 10,000 eggs/100 cm³ of soil in 15-cm depth. At transplanting of 4-week old watermelon seedlings, soils were sampled for the bioassay or for extraction of J2 by Baermann funnel. In the Seinhorst function of harvested biomass in relation to nematode numbers, decline of biomass with increasing population densities of M. incognita was accurately modeled by the inoculated eggs (R² = 0.93) and by the counts of galls on the bioassay roots (R² = 0.98); but poorly by J2 counts (R² = 0.68). Threshold levels of watermelon top dry weight to M. incognita were 122 eggs/100 cm³ soil, 1.6 galls on bioassay roots, or 3.6 J2/100 cm³ of soil. Using the bioassay in early spring for predicting risk of nematode damage appeared useful in integrated pest management systems of watermelon.     

Persistence of Heterorhabditis Infective Juveniles in Soil: Comparison of Extraction and Infectivity Measurements

The persistence of Heterorhabditis megidis in soil was studied over a 4-week period. On days 0, 2, 14, and 28, infective juveniles (IJ) were extracted by centrifugal flotation, Baermann funnel, and baiting of soil with Tenebrio molitor larvae, which were then dissected. Extraction efficiencies on day 0 were 82% by centrifugal flotation, 56% by Baermann funnel, and 19.8% by bait insect. The relative efficiency of the three methods changed over time. The relationship between the density of nematodes in the soil and the proportion recovered by dissection was non-linear. Up to a dose of approximately 60 IJ/insect, less than 12% became established, while at higher doses (up to 200 IJ/insect) the invasion efficiency was 23%. Mortality of bait insects increased from day 0 to day 2, but decreased to day 28. A novel method of assessing soil pathogenicity by preparing a soil density series and calculating the dose of soil or IJ that kills 50% of the bait insects gave a similar pattern. This method is recommended as a means of tracking changes in pathogenicity over time when bait insect mortality in undiluted soil is at or near 100%. Two methods of preparing a series of Heterorhabditis IJ densities in soil, either by diluting the soil itself with IJ-free soil or by adding diluted suspensions of IJ to the soil, resulted in the same bait insect mortalities.     

捕食线虫真菌对松材线虫捕食率测定的新方法

本文依据捕食线虫真菌的捕食特性以及松材线虫的生长习性,对松材线虫进行了定时,定量的测定试验,试图通过培养后线虫的种群数量变化,来摸索定量测定捕食率的方法.在漏斗法基础上进行了相应的改进,成功地摸索出3种方法捕食率测定的新方法.各方法各具优缺点,滤膜镜检法和双层镜检法的数据更精确,条切镜检法和漏斗法所测数值较粗略.因此,可依据实验的侧重点以及实验所需的精密程度,而采用最适宜的方法进行捕食率测定实验.     

茂兰喀斯特森林不同地形植物多样性与土壤理化特征研究

为探讨不同地形植物群落物种多样性与土壤理化特征之间的相互关系。该文以茂兰喀斯特森林为研究对象,分析了不同地形植物多样性与土壤理化特征以及两者之间的相互关系。结果表明:(1)不同地形木本植物的物种组成存在差异,坡地木本植物有35科65属78种,槽谷木本植物有38科64属89种,漏斗木本植物有35科61属84种。同时,丰富度指数、多样性指数、均匀度指数大小表现为槽谷漏斗坡地;优势度指数大小表现为坡地漏斗槽谷。(2)不同地形土壤物理性质差异性显著(P0.05),其中土壤容重和非毛管孔隙度表现为坡地槽谷漏斗,自然含水量、田间持水量、总孔隙度、毛管孔隙度等指标均表现为漏斗槽谷坡地。(3)除全K外,大多数土壤养分指标表现为漏斗显著高于槽谷、坡地,即漏斗槽谷坡地。(4)冗余分析表明,植物多样性与土壤理化性质具有相关性,不同地形植物多样性指数受土壤理化性质的影响明显。以上结果旁证了茂兰喀斯特森林地形条件的复杂性和土壤理化性质的差异性是该区小生境多样、物种组成丰富的原因之一,为喀斯特森林物种多样性维持机制提供了科学依据。     

Microfauna associated with algal mats in melt ponds of the Ross Ice Shelf

Summary The microfauna of benthic algal mats in eight ponds of diverse chemistry on the Ross Ice Shelf was examined. Tardigrades, nematodes and rotifers were successfully extracted from algal material by a modified Baermann funnel technique, which was robust enough to use under field conditions. Seven species of rotifer (of which 6 were bdelloids) and one species of nematode and tardigrade were found, all of which are cosmopolitan to Antarctic lentic ecosystems. Philodina sp. was the only invertebrate to occur in all ponds sampled. Rotifers were the most abundant invertebrates with average densities in all ponds of 4.26×10⁵ individuals/m², whereas nematodes were least abundant with densities of 4.96×10⁴ individuals/m². Total invertebrate densities and densities of individual taxa were significantly different in each of the 8 ponds, and of those measured environmental parameters were most strongly associated with conductivity. Cores taken from four ponds 2 cm into underlying substratum revealed that most animals live within the algal mats, with underlying sediment supporting a minimal population.     

The Baermann technique for estimating Protostrongylus infection in bighorn sheep: effect of laboratory procedures

The modified Baermann funnel technique was evaluated to determine the effects of time of baermannization, fecal preparation, type and size of funnel, and type of filter on the number of first stage larvae of Protostrongylus spp. recovered from feces of Rocky Mountain bighorn sheep (Ovis canadensis). More larvae were recovered when fecal pellets were baermannized for 24 hr compared to 8 hr, and when feces were crushed than when left intact. Use of small funnels resulted in the recovery of more larvae per gram of feces than larger funnels, and glass funnels more than plastic ones. There was no difference in recovery of larvae between cheesecloth filters and cellulose filters.     

Dynamics of the Entomogenous Nematode Steinernema feltiae Applied to Soil with and without Nematicide Treatment

The dynamics of Steinernema feltiae strain DD-136 in soils with different fauna was investigated to determine the best method for the biological control of soil insects. Infective juveniles (J3) were applied to field plots with and without 1,3-D (Telone II) fumigation. Recovery of J3 and changes in native nematode fauna were monitored until the applied J3 were no longer recovered by Baermann funnel (BF). Recovery of J3 by BF or by a two-step extraction procedure from steam-sterilized or nonsterilized sandy or silty soil with different fauna was investigated. More DD-136 J3 were recovered from the 1,3-D treated soil than from nontreated soil, while native nematodes in the treated soil fluctuated more with the addition of DD-136 than those in nontreated soil. The J3 persisted longer in silty than in sandy soils. The inundative soil application of DD-136 increased native rhabditids and decreased plant-parasitic nematodes. DD-136 in chemically treated soil not only effectively attacked the invading soil insect pests but also suppressed the recovery of plant nematodes.     

Arbuscular fungi and mycorrhizae (Glomales) of the Hel Peninsula,Poland

In the years 1985–1989, the occurrence of arbuscular fungi and mycorrhizae on the Hel Peninsula (Poland) was investigated with the help of 45 soil and root samples collected under 20 plant species of eight families. Except for Zea mays, the other plant species were from uncultivated sites. All soil samples contained spores of arbuscular fungi, of which about 45% were of the genus Glomus. Acaulospora spp. preferred members of the Cupressaceae. Spores of Gigaspora occurred rarely and only in two plant families. Glomus spp. were most frequently associated with plants of the Rosaceae, and species of Scutellospora were found at markedly higher frequencies among roots of plants of the Gramineae and Cupressaceae. A total of 29 spore-forming species and Glomus tenue (a fungus recognizable by its distinctive infections) were found. The most frequently recovered fungus, Glomus tenue, was present in roots of 56.8% of examined plants. Of the spore-forming fungi, the most frequently isolated spores were those of Scutellospora dipurpurascens, then Glomus constrictum, Acaulospora 61, and Glomus microcarpum. The overall spore density in examined samples averaged 99.8 in 100 g dry soil in the range 1 to 547, and was highest in a sample taken from around roots of Festuca arundinacea. The dominant fungi forming spores in sampled soils were Glomus constrictum, Glomus microcarpum, and Scutellospora dipurpurascens. The average species density was 3.9 in 100 g dry soil in the range 1 to 10, and was highest in Corynephorus canescens, Rosa canina, and Thuja occidentalis. Levels of colonization by arbuscular fungi ranged from 0.0 to94.0% (mean 23.3%) of the root length and were highest in Festuca arundinaceae and Zea mays.     

Inoculation with remnant prairie soils increased the growth of three native prairie legumes but not necessarily their associations with beneficial soil microbes

Restoring the diversity of plant species found in remnant communities is a challenge for restoration practitioners, in part because many reintroduced plant species fail to establish in restored sites. Legumes establish particularly poorly, perhaps because they depend on two guilds of soil microbial mutualists, rhizobial bacteria and arbuscular mycorrhizal (AM) fungi, that may be absent from restored sites. We tested the effect of soil microorganisms from remnant and restored prairies on legume growth by inoculating seedlings of Lespedeza capitata, Amorpha canescens, and Dalea purpurea with soil from 10 restored prairies and 6 remnant (untilled) prairies from southwest Michigan. We generally found support for the hypothesis that restored prairie soils lack microbes that enhance prairie plant growth, although there was variation across species and mutualist guilds. All three legumes grew larger and two legumes (Lespedeza and Amorpha) produced more nodules when inoculated with soil from remnant prairies, suggesting that low quantity and/or quality of rhizobial partners may limit the establishment of those species in restored prairies. In contrast, no legume experienced greater root colonization by AM fungi in remnant prairie soils, suggesting equivalent quantity (but not necessarily quality) of fungal partners in remnant and restored prairie soils. We detected no evidence of spontaneous recovery of the community of beneficial soil microbes in restorations. These results suggest that the absence of rhizobia, a largely overlooked component of prairie soils, could play a strong role in limiting restored prairie diversity by hindering legume establishment. Active reintroduction of appropriate rhizobial strains could enhance prairie restoration outcomes.     

Accelerated Movement of Nematodes from Soil in Baermann Funnels with Temperature Gradients

Baermann funnels were modified to eliminate or reverse the small temperature gradient (1-2 C/cm) across the soil layer that normally results from water evaporation. Effects of modifications on extraction efficiency were examined at various ambient temperatures and after overnight adaptation of three nematode species at 20 and 30 C. Extraction of Meloidogyne incognita from sandy loam, Tylenchulus semipenetrans from sandy clay loam, and Rotylenchulus reniformis from silt was greatly accelerated simply by covering funnels to prevent evaporation. In most cases, covering increased the nematodes extracted by 10-100 times after 5.5-48 hours. Faster and more efficient extraction of R. reniformis occurred over a wide range of ambient temperature (18-29 C). Effects of ambient temperature and temperature gradient direction on Baermann funnel extraction of R. reniformis were partly inconsistent with the behavior of R. reniformis in agar. Nematodes in agar moved toward cold at some ambient temperatures and toward heat at other temperatures. They always appeared to move toward cold on Baermann funnels. Differences were not attributable to blockage of gas exchange by covers. In agar and in funnels, the patterns of response to ambient temperature were shifted in the direction of the storage temperature.     

Primary Succession of Soil Rotifers in Clays of Brown Coal Post‐Mining Dumps

Changes in rotifer soil communities along a primary succession chronosequence was studied on brown coal post mining areas near Sokolov, NW part of the Czech Republic. The chronosequence of unreclaimed plots was 2, 11, 14, 20, 43 years old. The rotifers were extracted from soil samples using a modification of the Baermann funnel method with combined light and temperature gradients. In total, 34 taxa of soil rotifers were identified throughout the study. The most common species were Encentrum arvicola, Adineta vaga, A. steineri, Habrotrocha rosa, H. elegans, H. filum, Macrotrachela quadricornifera and M. nana. Rotifer abundance varied from 4 ± 2 · 10³ to 516 ± 488 · 10³ individuals m^–2. Species number per sample increased with age of the plot (r = 0.45, P = 0.003). The most important environmental variables which significantly affected rotifer community were wood cover, sodium concentration and age of the plot. Pioneer plant species occupied 2 and 11 year old plots, 14–20 year old plots were covered by Salix caprea shrubs and a forest formed by Betula pendula and Populus tremuloides developed on the 43 year old plot. Some species were ubiquitous and present throughout the chronosequence (Macrotrachela quadricornifera). Among the pioneer species were Encentrum incisum, Habrotrocha rosa and Macrotrachela papillosa, 14–20 years old plots were preferred by Adineta vaga, E. arvicola, H. filum and M. nana, while the oldest plot was dominated by Adineta steineri and Encentrum mucronatum. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Recovery of mycorrhizas of a fungus, <Emphasis Type="Italic">Cenococcum geophilum</Emphasis>, after urea treatment in warm temperate forests in Japan

We studied recovery of the ectomycorrhizal species Cenococcum geophilum (Cg) after urea treatment in two types of vegetation. The recovery was as quick as 6 months after the treatment and overlapped the fruiting period of ectomycorrhizal ammonia fungi. Ectomycorrhizas investigated as Cg belonged to a single species irrespective of treatment and vegetation type. Cg ectomycorrhizas were significantly more abundant in density in nontreated soil than in treated soil. Between vegetation types, Cg ectomycorrhizas were significantly more in density in broad-leaved deciduous forest than in broad-leaved evergreen forest. Moreover, Cg was dominant in the nontreated soils of the former type of forest.     

科尔沁沙地樟子松人工林土壤真菌共现网络及其与土壤因子的关系

为揭示科尔沁沙地樟子松(Pinus sylvestris var.mongolica)人工林土壤真菌间相互作用,以不同林龄(中龄、近熟和成熟)、不同土层(0—10和10—20 cm)樟子松人工林土壤为研究对象,以未造林地为对照样地,采用分子生态网络分析法,基于随机矩阵理比较分析樟子松人工林土壤真菌网络特征及其影响因素。研究结果表明：(1)中龄林到近熟林阶段,科尔沁沙地樟子松人工林土壤真菌网络平均连通度增大,平均路径长减小,网络愈加复杂,而近熟林到成熟林阶段则相反,网络愈加简单;不同土层间,10—20 cm土层土壤真菌平均路径长度小于0—10 cm土层,平均连通度和聚类系数大于0—10 cm土层,10—20 cm较0—10 cm土壤真菌间相互联系更为密切;与樟子松人工林相比,未造林地土壤真菌网络中的平均连通度略低于近熟林,平均路径长低于樟子松人工林,未造林地土壤真菌群落更不稳定。(2)中龄、近熟和成熟樟子松人工林土壤真菌关键菌种分别为Guehomyces sp.、Oidiodendron sp.和Pseudeurotium sp.,未造林地关键菌种为Alphamyces sp.。在0—1...     

Studies on pathogenic dematiaceous fungi

Two hundred and twenty-six samples of woody materials, vegetable matter and soil were processed by the direct plating and mouse inoculation technique for the isolation of pathogenic dematiaceous fungi. The species of fungi isolated were Fonsecaea pedrosoi — 13, Cladosporium carrionii — 7 and Phialophora verrucosa — 4 isolates. The mouse inoculation technique was found to be much better than direct plating for the recovery of these fungi. Woody plant materials proved to be a good sample source for pathogenic dematiaceous fungi contributing about 90% of the isolates. All the isolates were pathogenic for mice as evidenced by the presence of dark nodular lesions containing fungal elements in the organs of experimentally infected animals.     

Soil mycoflora from trufflè native areas of Saudi Arabia

Soil fungi of areas in the North-Eastern region of Saudi Arabia where truffles are native were surveyed. Forty-three species of fungi belonging to twenty genera were isolated. Most were recovered from soils underneath or around truffle ascocarps: thirty species from soil under the surface of Tirmania nivea ascocarps, twenty-four from Terfezia boudieri soil and twenty species each from Tirmania pinoyi and Terfezia claveryi soils. Rhizosphere soil of Helianthemum lippi, on the other hand, yielded twenty-four fungal species while only fourteen fungal species were found in soil without vegetation. The total counts of fungi/g soil were highest in soils from the under surface of truffles, followed by rhizosphere soil, with the lowest in soils without vegetation.