Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

北京地区苹果及葡萄根际捕食线虫真菌的种类及分布

对北京郊区果园的苹果和葡萄根际的捕食线虫真菌进行调查,发现苹果和葡萄的根际不同位置的捕食线虫真菌数量不同,内根际（包括根表）捕食线虫真菌数量大于外根际,而外根际捕食线虫真菌数量又大于游离土壤中的捕食线虫真菌数量;在苹果根际发现的捕食线虫真菌共有９种：Ａｒｔｈｒｏｂｏｔｒｙｓｄａｃｔｙｌｏｉｄｅｓ,Ａ．Ｃｌｉｇｏｓｐｏｒａ,Ａ．Ｍｕｓｉｆｏｒｍｉｓ,Ａ．Ｃｌａｄｏｄｅｓ,Ａ．Ｂｒｏｃｈｏｐａｇａ,Ａｒｔｈｒｏｂｏｔｒｙｓｓｐ．,Ｍｏｎａｃｒｏｓｐｏｒｉｕｍｅｕｄｅｒｍａｔｕｍ,Ｍ．Ｓｐｈ     

Identification of fungi associated with rotylenchulus reniformis

The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.     

捕食线虫真菌在北京地区果园中的季节性分布

通过对北京海淀区2个苹果园和2个葡萄园根际捕食线虫真菌2年的季节性变化调查,共分离到199个捕食线虫真菌菌株,隶属3个属16个种,Arthrobotrysdactyloides分离频率最高,其次是A.oligospora,A.conoides和Stylopagesp.。捕食线虫真菌的数量在晚春夏初最丰富,秋季次之,冬夏最少。通过统计分析,捕食线虫真菌数量的季节性变化与温度线性相关,而与线虫总数S形相关,与土壤湿度和植物寄生线虫数量无显著相关。     

Predacity by nematophagous fungi and its relation to the attraction of nematodes

Predacity, the ability of nematophagous fungi to destroy nematodes, was investigated for eight species of fungi by a method using sterilized soil and the nematodePanagrellus redivivus. In addition, the ability of the fungi to attract nematodes was investigated using an agar plate technique. Predacity and attraction were highly correlated (r=0.98) in these tests. The presence of traps in cultures ofArthrobotrys oligospora increased the ability to attract nematodes by a factor of 2.     

The living strategy of nematophagous fungi

The infection structures, trophism, and ecological character of nematophagous fungi are reviewed in this article on the basis of data extracted from the literature and the most recent experiments conducted in this area. Traditionally, nematophagous fungi are classified into four groups according to their modes of attacking nematodes: nematode-trapping fungi using adhesive or mechanical hyphal traps, endoparasitic fungi using their spores, eggparasitic fungi invading nematode eggs or females with their hyphal tips, and toxin-producing fungi immobilizing nematodes before invasion. In the present review, we focus on the first two groups. The living strategies of these nematophagous fungi depend on the diversity of their infection structures, such as different traps and spore types, which determine the modes of infecting nematodes. The diversity of trophic modes of nematophagous fungi is an important prerequisite for fungal survival and activity in soil. The abundance and activity of Hirsutella rhossiliensis and H. minnesotensis, representatives of endoparasites and potential biocontrol agents against nematodes, are highly dependent on environmental factors. Comprehensive understanding of the survival and activity of nematophagous fungi in soil is fundamental for the exploitation of these fungi as successful biocontrol agents.     

Extracellular enzymes and the pathogenesis of nematophagous fungi

Nematophagous fungi are an important group of soil microorganisms that can suppress the populations of plant-parasitic nematodes. The pathogenic mechanisms of nematophagous fungi are diverse: They can be parasitical–mechanical through producing specialized capturing devices, or toxin-dependent. During infections, a variety of virulence factors may be involved against nematodes by nematophagous fungi. In this review, we present up-to-date information on the modes of infection by nematophagous fungi. The roles of extracellular hydrolytic enzymes and other virulence factors involved in infection against nematodes were summarized. The biochemical properties and peptide sequences of a special group of enzymes, the serine proteases, were compared, and their implications in infections were discussed. We also discussed the impact of emerging new techniques on our understanding of this unique group of fungi.     

First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica

A survey of entomopathogenic nematodes was conducted in the north Pacific (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identified as Heterorhabditis indica and the three Steinernema isolates were identified as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing.     

食线虫真菌致病相关丝氨酸蛋白酶的研究进展

食线虫真菌是一类土壤微生物,作为线虫的天敌,它们对于维持线虫在土壤生态环境中的种群动态平衡发挥着十分重要的作用。食线虫真菌通过形成特殊的捕食器官或产生毒素等方式来捕捉和杀死线虫。丝氨酸蛋白酶是食线虫真菌侵染线虫的重要毒力因子,近年来,研究人员对不同食线虫真菌来源的致病相关丝氨酸蛋白酶进行了大量的研究,尤其在丝氨酸蛋白酶的晶体结构和分子进化方面的研究取得了较大的进展。本文对食线虫真菌致病相关丝氨酸蛋白酶的生物化学性质和功能进行了系统的总结,对丝氨酸蛋白酶的晶体结构、催化机制及分子进化等最新的进展进行了评述。     

Nematophagous fungi: Endoparasites ofRhabditis terricola

Three techniques were compared for the recovery of endoparasitic nematophagous fungi from soil. The Baermann funnel technique (32 species) was superior to differential centrifugation (19 species) or soil sprinkling (21 species) in terms of number of species found. The main advantage of the Baermann funnel was in the recovery of lower fungi, especially those with flagellate states. In all, 40 species of endoparasites (70% of the known flora) were recorded. Thirty-two of those were found capable of attacking the soil nematodeRhabditis terricola Dujardin.     

Control of Root-Knot Nematodes on Tomato by the Endoparasitic Fungus Meria coniospora

The endoparasitic nematophagous fungus Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trials. The fungus was introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematodes infected with M. coniospora to the soil; both methods reduced root galling by root-knot nematodes. These studies represent a part of a recently initiated effort to evaluate the potential of endoparasitic nematophagous fungi for biocontrol of nematodes.     

根结线虫天敌真菌的筛选研究初报

从土壤和病株上分离到10个线虫的天敌真菌菌株,其中有三个菌株较有希望,烛台霉属(Candelabrello sp.)一个。孤孢属(Monacrosporium spp.)两个,菌株代号分别为CN_7;CN_(?)和CN_5。 据试验:CN_7较好,菌丝体生长的温度范围是20～33℃,最适宜的温度范围是25～30℃,以式捕捉环捕食线虫,对培养基选择性不强,最适宜的pH值是6.0～7.5,但在pH4.5～8.0都能进收缩行捕捉,捕捉器官形成量多,捕虫势强且稳定,在水中也能形成捕捉环并捕食线虫。     

A bird's eye survey of Central American planorbid molluscs

In the course of two trips to Central America (June 1967 and JulyAugust 1976) I had the opportunity of collecting topotypic specimens of Planorbis nicaraguanus Morelet, 1849, anatomically defined in this paper, and of P. yzabalensis Crosse & Fischer, 1879, the identity of the latter with Drepanotrema anatinum (Orbigny, 1835) is confirmed. The following planorbid species were also found: Helisoma trivolvis (Say, 1817) in Nicaragua, Guatemala, Costa Rica and Belize; H. duryi (Wetherby, 1879) in Costa Rica; Biomphalaria helophila (Orbigny, 1835) in Guatemala, Belize, Nicaragua, Costa Rica and El Salvador; B. kuhniana (Clessin, 1883) in Panama; B. obstructa (Morelet,1849) in Guatemala, Belize and El Salvador; B. straminea (Dunker, 1848) in Costa Rica; B. subprona (Martens, 1899) in Guatemala; D. anatinum (Orbigny,1835) in Belize, Guatemala, Nicaragua and Costa Rica; D. depressissimum (Moricand,1839) in Nicaragua, Costa Rica and Panama; D. lucidum (Pfeiffer, 1839) in Guatemala, Belize and Nicaragua; D. surinamense (Clessin, 1884) in Costa Rica and Panama; and Gyraulus percarinatus sp. n. in Panama. The occurrence of B. kuhniana and D. surinamense is first recorded in Central America, and Gyraulus percarinatus is the first representative of the genus provenly occurring in the American continent south of the United States. The following synonymy is proposed: Planorbis declivis Tate, 1870 = Biomphalaria helophila (Orbigny, 1835); Planorbis isthmicus Pilsbry, 1920 = Biomphalaria kuhniana (Clessin, 1883); Planorbis cannarum Morelet, 1849 and Segmentina donbilli Tristram, 1861 = Biomphalaria obstructa (Morelet, 1849); and Planorbis yzabalensis Crosse & Fischer, 1879 = Drepanotrema anatinum (Orbigny, 1835), confirming Aguayo (1933).     

Addendum to the checklist of the helminth parasites of vertebrates in Costa Rica

In this work, we present an addendum to the "Checklist of the helminth parasites of vertebrates in Costa Rica" with a parasite-host list as well as a host-parasite list. This addendum updates the available information on this group of parasites in Costa Rica, since very recently a new input has been made to describe the helminth fauna of vertebrates, particularly at the Area de Conservación de Guanacaste. In this paper, we add 33 records, representing 23 species. This raises the number of helminth species described in vertebrates from Costa Rica to 325, represented by 89 species of digeneans, 23 of monogeneans. 63 of cestodes, 13 of acanthocephalans, and 137 of nematodes. In total, 133 species of vertebrates have been studied for helminths in Costa Rica (31 species of fishes. 7 amphibians, 18 reptiles, 40 birds, and 37 mammals). Currently, 67 species (20.6 %) have been recorded as new species from Costa Rica and most of them are endemic to particular regions. The Colecci6n de Helmintos de Costa Rica, housed at the Universidad de Costa Rica (UCR), San José, is the depositary of only 23% of the species recorded so far; however this situation is changing and people recognizes.     

The effect of replication on the probability of isolating nematophagous fungi from soil

The exact number of replicates required for a given probability of isolating all the species of nematophagous fungi present in a single sample of soil was measured. Using the soil sprinkling technique twenty replicate sub-samples of a sample of farm soil were examined for species. Eight replicates were required under these circumstances to be 95% sure of isolating all the species present. An equation to calculate the number of replicates to achieve a given probability of 100% species isolation is also given.     

Suppression of nematophagous fungi by enchytraeid worms: a field exclosure experiment

The feeding biology of Enchytraeus crypticus and other enchytraeids is poorly understood as is their effect on nematophagous fungi. Because enchytraeids had been associated with nematophagous fungi in the field and had suppressed these fungi in soil microcosms, we tested the hypothesis that exclusion of enchytraeids, largely E. crypticus, would improve establishment of certain nematophagous fungi in field plots. The fungi, Hirsutella rhossiliensis and Monacrosporium gephyropagum, are being studied as potential control agents of plant-parasitic nematodes and were formulated as hyphae in alginate pellets. The pellets were mixed into soil without enchytraeids and placed in cages (PVC pipe, 80 cm³ volume) with fine (20 μm) or coarse (480 μm) mesh; cages were buried 15 cm deep in field plots and then recovered after 6–52 days. When fine mesh was used, enchytraeids were excluded and the fungi increased to large numbers. When coarse mesh was used, enchytraeid numbers in cages increased rapidly and the fungi did poorly. Although mesh also affected other potential fungivores, including collembolans and large dorylaimid nematodes, we suspect that enchytraeids were more important because large numbers were consistently found in cages with coarse mesh soon after the cages were placed in soil. Organisms smaller than enchytraeids (bacteria, fungi, and protozoa) also appeared to be important because the fungi did better in heat-treated soil than in non-heat-treated soil, regardless of mesh size. The rapid increase in enchytraeid numbers in cages with hyphal pellets and coarse mesh was probably caused by movement of enchytraeids toward the pellets with hyphae: increase in enchytraeid numbers was minimal when movement into cages was blocked (or when cages contained pellets without hyphae). Overall, the data were consistent with the hypothesis that enchytraeids, or other meso- or macrofauna, contributed to suppression of nematophagous fungi in our field plots. Received: 22 April 1997 / Accepted: 16 June 1997     

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

Abstract The nematophagous fungi ( Arthrobotrys oligospora and Monacrosporium cionopagum ) were observed under semi-natural conditions by fluorescence microscopy after fluochroming, using fluorescein-diacetate. The formation of 3-dimensional sticky networks and the capture of nematodes inside the soil could be demonstrated.     

In vitro soil receptivity assays to egg-parasitic nematophagous fungi

Soil application of nematophagous fungi for the biological control of plant-parasitic nematodes often fails, and in many cases it has been difficult to reisolate the agent delivered to the soil. A reason for these results could be the inability of the fungi to proliferate in soil. We used a soil–membrane technique to study the capacity of several isolates of the nematophagous fungi Pochonia chlamydosporia and Paecilomyces lilacinus to grow and establish in sterilized and nonsterilized sandy soils from SE Spain and Western Australia. Growth of all fungi tested was inhibited in nonsterilized soil, although there was intraspecific variability in sensitivity among isolates of the same species. With respect to hyphal density, P. chlamydosporia isolate 5 (from Italy) was the least inhibited in nonsterilized soil from both sites. Relative growth analyses confirmed this result for soil from SE Spain, while with this method, P. chlamydosporia isolate 4624 (from Australia) appeared to be least inhibited in the Australian soil. The results indicate that a soil can be more receptive to its indigenous isolates than to nonindigenous isolates. Apparently, soil microbiota can determine the ability of nematophagous fungi to proliferate in soil.     

Checklist of the helminth parasites of vertebrates in Costa Rica

Helminth parasites of vertebrates have been studied in Costa Rica for more than 50 years. Survey work on this group of parasites is far from complete. We assembled a database with all the records of helminth parasites of wild and domestic vertebrates in Costa Rica. Information was obtained from different sources such as literature search (all published accounts) and parasite collections. Here we present a checklist with a parasite-host list as well as a host-parasite list. Up to now, 303 species have been recorded, including 81 species of digeneans, 23 monogeneans, 63 cestodes, 12 acanthocephalans, and 124 nematodes. In total, 108 species of vertebrates have been studied for helminths in Costa Rica (31 species of fishes, 7 amphibians, 14 reptiles, 20 birds, and 36 mammals). This represents only 3.8% of the vertebrate fauna of Costa Rica since about 2,855 species of vertebrates occur in the country. Interestingly, 58 species (19.1 %) were recorded as new species from Costa Rica and most of them are endemic to particular regions. Considering the valuable information that parasites provide because it is synergistic with all the information about the natural history of the hosts, helminth parasites of vertebrates in Costa Rica should be considered within any initiatives to accomplish the national inventory of biological resources. Starting with this compilation work, the Colección de Helmintos de Costa Rica (CHCR), hosted at the Facultad de Microbiología, Universidad de Costa Rica, has re-emerged and it is our hope that it will have the standards of quality to assure that it will become the national depository of helminths in the country.     

Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes

Efficient selection of fungi for biological control of nematodes requires a series of screening assays. Assessment of genetic diversity in the candidate species maximizes the variety of the isolates tested and permits the assignment of a particular genotype with high nematophagous potential using a rapid novel assay. Molecular analyses also facilitate separation between isolates, allowing the identification of proprietary strains and trace biocontrol strains in the environment. The resistance of propagules to UV radiation is an important factor in the survival of a biocontrol agent. We have analyzed 15 strains of the nematophagous fungus Paecilomyces lilacinus using these principles. Arbitrarily primed DNA and allozyme assays were applied to place the isolates into genetic clusters, and demonstrated that some genetically related P. lilacinus strains exhibit widespread geographic distributions. When exposed to UV radiation, some weakly nematophagous strains were generally more susceptible than effective isolates. A microtitre tray-based assay used to screen the pathogenic activity of each isolate to Meloidogyne javanica egg masses revealed that the nematophagous ability varied between 37%-100%. However, there was no clear relationship between nematophagous ability and genetic clusters. Molecular characterizations revealed sufficient diversity to allow tracking of strains released into the environment.