Association of Nematodes and Dogwood Cankers

Dogwood canker is a serious production problem of unknown etiology. From May 1985 through April 1989, cankers from 290 flowering dogwood trees in 15 separate nurseries were sampled for nematodes. Seventy-three percent (213) of the cankers contained nematodes. Panagrolaimus rigidus (Schneider) Thorne (115/290) and Aphelenchoides spp. (91/290) were the most frequently collected taxa. Panagrolaimus rigidus was reared on 2% water agar with unidentified bacteria as the food source. Aphelenchoides spp. were reared in antibiotic-amended agar culture with the fungus Glomerella cingulata (Stoneman) Spauld. &Schrenk as a food source. Repeated attempts to culture Aphelenchoides spp. on dogwood callus tissue were unsuccessful. Artificially created stem wounds inoculated with combinations of Aphelenchoides spp. and P. rigidus callused completely in 60 days with no indication of canker development. Very low numbers of nematodes were recovered from inoculated trees, but P. rigidus and one Aphelenchoides sp. were efficient dispersers and occurred in treatments other than those in which they were inoculated.     

Aphelenchoides hylurgi as a Carrier of White, Hypovirulent Cryphonectria parasitica and its Possible Role in Hypovirulence Spread on Blight-Controlled American Chestnut Trees

Individual nematodes were isolated from American chestnut blight-controlled cankers to determine if they were carriers of biocontrol (hypovirulent) isolates of the chestnut blight fungus, Cryphonectria parasitica. These hypovirulent isolates have a white fungal colony phenotype due to infection by the virus CHV1. Of 1,620 individual Aphelenchoides hylurgi isolated, 29.4% carried propagules of the blight fungus and 8.2% of these yielded white hypovirulent isolates. In attraction and movement tests in Petri plates, A. hylurgi moved 2 cm over 24 hr to mycelial discs of white hypovirulent C. parasitica and pigmented C. parasitica strains in nearly equal numbers. After 2 days of nematode movement to fungal colonies on agar in Petri plates and 21 days of nematode growth, large numbers of A. hylurgi were extracted from both white hypovirulent and pigmented C. parasitica strain colonies. Lower numbers of A. hylurgi were extracted from excised young American chestnut blight cankers that were inoculated with A. hylurgi and incubated for 22 days. A. hylurgi inoculated on the surface of an excised American chestnut canker moved within 24 hr to the small, spore-bearing C. parasitica reproductive structures (stromata) on the canker surface. The results indicate that A. hylurgi may play a role in the spread of hypovirulence on American chestnut trees.     

Inhibition of virulent and hypovirulent Cryphonectria parasitica growth in dual culture by fungi commonly isolated from chestnut blight cankers

Chestnut blight cankers, caused by the fungus Cryphonectria parasitica, are prone to invasion by other microorganisms as the canker ages. This microbial community has the potential to alter canker expansion, which may influence the probability that the canker girdles the infected stem. Hypoviruses infect the pathogen mycelium directly and are known to decrease pathogen virulence (i.e. hypovirulent). These viral infections can slow pathogen growth, decreasing the rate of canker expansion and lowering the probability of girdling. Saprophytic fungi also invade the expanding canker and may antagonize C. parasitica leading to reduced pathogen growth. The combined effects of fungal antagonism and a hypovirulent pathogen could work in combination to reduce the probability of girdling the infected stem. We assessed the ability of different fungal taxa, isolated from low severity cankers, to inhibit the growth of virulent and hypovirulent forms of C. parasitica in dual culture tests on two cultural media. Percent growth inhibition of virulent C. parasitica by potentially antagonistic fungi ranged from 2 % to 34 %, while inhibition of hypovirulent C. parasitica ranged from 18 % to 54 %. Only one isolate, identified as Umbelopsis isabellina (UmbelopsisWS) inhibited the virulent form of the pathogen more than the hypovirulent form. All three Trichoderma isolates caused the greatest growth inhibition of virulent C. parasitica, but they, like all other fungal isolates tested, inhibited the hypovirulent form of the pathogen more than the virulent form. These results suggest that commonly occurring fungi in chestnut blight cankers, including Trichoderma, may inhibit the hypovirulent C. parasitica more than virulent C. parasitica. Thus, the presence of other fungi in cankers may not enhance the effect of hypovirulent C. parasitica to delay cankers from girdling a stem but instead intensify canker development.     

Differential Transfer and Dissemination of Hypovirus and Nuclear and Mitochondrial Genomes of a Hypovirus-Infected Cryphonectria parasitica Strain after Introduction into a Natural Population

Biological control of plant diseases generally requires release of living organisms into the environment. Cryphonectria hypoviruses function as biological control agents for the chestnut blight fungus, Cryphonectria parasitica, and hypovirus-infected C. parasitica strains can be used to treat infected trees. We used naturally occurring molecular marker polymorphisms to examine the persistence and dissemination of the three genomes of a hypovirus-infected C. parasitica strain, namely, the double-stranded RNA genome of Cryphonectria hypovirus 1 (CHV1) and the nuclear and mitochondrial genomes of its fungal host. The hypovirus-infected strain was experimentally introduced into a blight-infested chestnut coppice forest by treating 73 of 246 chestnut blight cankers. Two years after introduction, the hypovirus had disseminated to 36% of the untreated cankers and to 35% of the newly established cankers. Spread of the hypovirus was more frequent within treated sprout clusters than between sprout clusters. Mitochondrial DNA of the introduced fungus also was transferred into the resident C. parasitica population. Concomitant transfer of both the introduced hypovirus and mitochondrial DNA was detected in almost one-half of the treated cankers analyzed. The introduced mitochondrial DNA haplotype also was found in three resident isolates from newly established cankers. The nuclear genome of the introduced strain persisted in the treated cankers but did not spread beyond them.     

Control of chestnut blight by the use of hypovirulent strains of the fungus Cryphonectria parasitica in northwestern Spain

Chestnut blight is controlled in Europe by using Cryphonectria hypovirus CHV1, a non-encapsulated RNA virus. The chestnut blight fungus, Cryphonectria parasitica, is weakened by the virus, and healing tissue growth occurs in the host tree. Transmission of this cytoplasmic hypovirus is restricted by the incompatibility system of the fungus, so that the hypovirus can be transmitted only between isolates of the same or closely related vegetative compatibility (vc) types. Hypovirulent isolates of C. parasitica (all of the French subtype CHV1-F1) from Castilla y León (NW Spain) were compared with virulent isolates in both laboratory (cut stems) and field inoculations (in two orchards in the province of León and one orchard in the province of Zamora). The tests were performed with the most common vc types in the region, EU1 and EU11. The cut stem assay revealed that the hypovirulent isolates of vc type EU1 did not reduce the growth of virulent cankers. By contrast, four hypovirulent strains H1, H4, H5 and H6 (all vc type EU11) reduced the growth of virulent isolates in the cut stem assay. Field tests showed that hypovirulent isolates of EU1 and EU11 were effective in reducing canker in both orchards in León with all treatments tested; however, in Zamora, where only EU11 was tested, all the treatments failed except H1, which was able to reduce growth of the canker eighteen months after the inoculation. The development of hypovirulence suggests that hypovirus subtype F1 is well adapted in the province of León. Both naturally extended and inoculated hypoviruses appear to have reduced the incidence of the canker, thus improving chestnut stands. However, the inoculations were not as effective in the orchards in Zamora. This indicates that the disease could be controlled in Castilla y León by inoculation of trees with hypovirulent strains, but that more tests should be done in provinces where the hypovirus is still not present.     

<Emphasis Type="Italic">Castanea sativa</Emphasis>: genotype-dependent recovery from chestnut blight

In Lovran (coastal Croatia), a unique forest/orchard of evenly mixed grafted marrons and naturally growing nongrafted sweet chestnut trees exists. This old chestnut population has been devastated by chestnut blight, caused by an aggressive introduced pathogenic fungus, Cryphonectria parasitica. However, initial observations indicated recovery of naturally growing chestnut trees in that area, mediated by Cryphonectria-associated hypovirus (Cryphonectria hypovirus 1 (CHV-1)). Such recovery was not observed on grafted trees. Genotyping both, we confirmed the clonal origin of the grafted ones—marrons. No significant difference was observed between fungal strains isolated from naturally growing trees and the ones from marrons regarding fungal vegetative compatibility types or the prevalence of CHV-1. A strong correlation was observed between the types of canker: active/deep-expanding versus healing callus or superficial necrosis and the absence or presence of CHV-1 in the fungal isolates, sampled from naturally growing trees (Spearman rho 0.686, p value 7.81?×?10^?5, Kendall tau 0.686, p value 5.18?×?10^?7). Such correlation was not observed on marrons (Spearman rho 0.236, p value 0.235, Kendall tau 0.236, p value 0.084), because, unexpectedly, active/deep-expanding cankers were often associated with hypovirulent fungal isolates. These data indicate that the lack or unequal distribution of naturally occurring hypovirulence were not the cause of substantial marron decay in Lovran. Ecological and age-dependant differences were ruled out because all sampled trees are growing in close proximity and are of similar age. The results imply that the marron genotype is especially vulnerable and its ability to recover is limited even when the hypovirulent strain of the fungus is present in the canker.     

Mass Production of the Mosquito Parasite Romanomermis culicivorax: Effect of Density

When numbers of Romanomermis culicivorax Ross and Smith were varied in containers with a constant surface area and depth of sand, densities of 12-24 nematodes per cm² yielded significantly more preparasites than higher densities. When container surface area and numbers of nematodes were constant and sand volume and depth were varied, yields did not differ significantly. When numbers of nematodes and sand volume were constant and surface area and sand depths were varied, yields were significantly higher for a density of 24 nematodes per cm². Yields of preparasites were tripled by simply setting up three cultures, each containing 5 g of nematodes, instead of a single culture containing 15 g.     

Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.     

Food attraction and population growth of fungivorous nematodes with different fungi

Food attraction of the fungivorous nematodes Aphelenchus avenae and Aphelenchoides spp. to seven fungal species (Pyrenochaeta lycopersici, Botrytis cinerea, Rhizoctonia solani strains AG 3 and AG 2‐1, Verticillium dahliae, Pochonia bulbillosa, Mortierella hyalina and Trichoderma harzianum) was determined on agar plates by counting the number of test nematodes present on the mycelium of each fungus 24 h after inoculation. Population growth of A. avenae and Aphelenchoides spp. on five of the seven fungi included in the attraction test (P. lycopersici, R. solani strain AG 3, V. dahliae, P. bulbillosa and T. harzianum) was also determined on agar plates by counting nematode numbers every week during a 6‐week period. A. avenae and Aphelenchoides spp. were attracted to all the fungi tested. A. avenae was preferentially attracted to V. dahliae (P < 0.0001), and Aphelenchoides spp. did not show any preference except for low attraction to R. solani. A. avenae and Aphelenchoides spp. reproduced on all fungal species tested. After 6 weeks of incubation, the highest number of nematodes was found on P. lycopersici and P. bulbillosa, while the lowest number occurred on R. solani for A. avenae and on T. harzianum for Aphelenchoides spp. The suitability of a fungus as a host was not clearly related to the attraction to that fungus.     

Morphological,molecular and biological characterization of Esteya vermicola,a nematophagous fungus isolated from intercepted wood packing materials exported from Brazil

The nematophagous fungus Esteya vermicola, strain NKF 13222, was purified from an isolate of Bursaphelenchus rainulfi which was intercepted from wood packaging materials originating in Brazil and arriving at Tianjin port in China. The fungus produced two types of conidiogenous cells and conidia, each with different germination modes. More lunate adhesive conidia than bacilloid conidia were produced on nutrient-poor water agar medium. Morphological comparisons revealed the NKF 13222 strain closely resembled the Taiwan strain E. vermicola (ATCC 74485) previously isolated from the pinewood nematode B. xylophilus. Phylogenetic analysis of the β-tubulin and elongation factor 1-α genes indicated that the NKF 13222 grouped with other strains of E. vermicola including the Taiwan strain. This was the first record of E. vermicola from B. rainulfi in South America. Infection tests demonstrated that NKF 13222 was more infective to aphelenchid than tylenchid nematodes and that only lunate adhesive conidia were infectious. The results suggest that the fungus might be a pathogen of plant parasitic nematodes with a broad distribution and provide new information for the potential biocontrol of plant diseases caused by B. xylophilus, Aphelenchoides spp. and Ditylenchus destructor.     

Abundance, distribution and feeding relations of root/fungal feeding nematodes in a Scots pine forest

In a 15–20 yr old forest of Pinus sylvestris, Tylenchus spp., Paratylemchus spp? and Malenchus tantulus were the most abundant stylet-bearing nematodes. Soil layers and ground cover affected the abundance. Mycophagy was observed for Tylenchus sp., M. tantulus, Aphelenchoides sp. and Tylencholaimus stecki. Feeding on roots of the forest grass, Deschampsia flexuosa was recorded for W. tantulus, Paratylenchus spp? and Rotylenchus sp. The latter fed also on pine roots. Connections occurred between feeding habits and field distribution. The facultative feeding habit of M. tantulus probably allowed this species to reach higher numbers under D. flexuosa. The importance, in the field, of the grass Calamagrostis arundinacea for Paratylenchus spp? was confirmed by laboratory experiments. As pine was a non-host, these nematodes were probably grass feeders. Paratylenchus spp? dominated the obligate root feeders with forest grass being a key-factor for the group abundance. Suspected high densities of Paratylenchus spp? on clearings should not affect the growth of pine seedlings and although Rotylenchus sp. to some extent reduced seedling growth its pathogenic status on P. sylvestris was doubtful.     

Mass Culture of Subanguina picridis and Its Bioherbicidal Efficacy on Acroptilon repens

A Russian knapweed (Acroptilon repens) shoot culture system, initiated from shoot tip culture, was used to generate a source of host plant tissue for the rearing of the nematode Subanguina picridis, a biocontrol agent for Russian knapweed. Young shoots growing on solid B5G medium in petri dishes developed galls on leaves, petioles, and shoot tips 7 days after release of 50 nematodes onto the surface of the medium. After 3 months of culturing, each petri dish yielded 7,000-10,000 nematodes. In vitro cultured Subanguina picridis were virulent on greenhouse-grown Russian knapweed plants. Galls were first found on seedlings 12 days after infestation; after 2 months, 90% of seedlings were galled on leaves, petioles, and shoot tips, with 1-6 galls per seedling. Three months after shoot emergence, 64% of vegetative shoots originating from root segments were also galled by the cultured nematodes. Similarly, vegetatively regenerated shoots of Russian knapweed were also susceptible to infestation by cultured nematodes.     

Additive Effects of Meloidogyne arenaria and Sclerotinin rolfsii on Peanut

Field observations have suggested that infection of peanut by Meloidogyne arenaria increases the incidence of southern blight caused by Sclerotium rolfsii. Three factorial experiments in microplots were conducted to determine if interactions between M. arenaria and S. rolfsii influenced final nematode population densities, incidence of southern blight, or pod yield. Treatments included four or five initial population densities of M. arenaria and three inoculum rates of S. rolfsii. Final nematode population densities were affected by initial nematode densities in all experiments (P = 0.01) and by S. rolfsii in one of three experiments (P = 0.01). Incidence of southern blight increased with increasing inoculum rates of S. rolfsii in all experiments and by the presence of the nematodes in one experiment (P = 0.01). Pod yield decreased with inoculation with S. rolfsii in all experiments (P = 0.05) and by M. arenaria in two of three experiments (P = 0.05). In no experiment was the interaction among treatments significant with respect to final nematode population densities, incidence of southern blight, or pod yield (P = 0.05). The apparent disease complex between M. arenaria and S. rolfsii on peanut is due to additive effects of the two pathogens.     

Distribution and dynamics of mycophagous and microbivorous nematodes in potato fields and their relationship to some food sources

Nematodes are not uniformly distributed in soil. The aim of this study was to investigate the distribution pattern of mycophagous and microbivorous nematodes and to develop sampling methods to detect them at different sites in the soil. Sampling of nematodes was carried out by three methods: using an auger, by collecting soil which was shaken from the roots (root shaking sample) and by collecting roots with the adhering rhizosphere soil (rhizosphere sample). Distribution patterns and population dynamics of mycophagous nematodes were similar to other microbivorous nematodes in three potato fields. Population densities in the rhizosphere samples were 4–50 times higher than those in auger samples. Samples obtained with the auger and root-shaking methods yielded more or less equal population densities. Numbers of the mycophagous nematodes Aphelenchus avenae, Aphelenchoides sp. and unidentified microbivorous nematodes increased several fold within a few days on suitable substrates in soil. These substrates could be the dying roots of plants of which the haulms were killed or flax straws that were previously colonised by the fungus Rhizoctonia solani.     

Effect of Hirschmanniella caudacrena on the Submersed Aquatic Plants Ceratophyllum demersum and Hydrilla verticillata

In vitro pathogenicity tests demonstrated that Hirschmanniella caudacrena is pathogenic to Ceratophyllum demersum (coontail). Symptoms were chlorotic tissue, deformed stems, and, finally, death of the plant. Inoculum densities of 500 nematodes per 5-cm-long cutting in a test tube containing 50 ml of water resulted in death and decay of some of the cuttings within 8 weeks; 100 nematodes killed the plants in 12 weeks, and 50 and 25 nematodes killed them in 16 weeks. The lowest inoculum level of 10 nematodes did not seriously affect the plants at 16 weeks when the experiment was terminated. A second test conducted outdoors in glass jars containing 3 liters of water and two cuttings weighing a total of 15 g fresh weight showed damage, but results were not statistically significant. Hydrilla verticillata inoculated with H. caudacrena was not affected seriously.     

Plant-parasitic Nematode Communities and Their Associations with Soil Factors in Organically Farmed Fields in Minnesota

A survey was conducted to determine the assemblage and abundance of plant-parasitic nematodes and their associations with soil factors in organically farmed fields in Minnesota. A total of 31 soil samples were collected from southeast (SE), 26 samples from southwest (SW), 28 from west-central (WC), and 23 from northwest (NW) Minnesota. The assemblage and abundance of plant-parasitic nematodes varied among the four regions. The soybean cyst nematode, Heterodera glycines, the most destructive pathogen of soybean, was detected in 45.2, 88.5, 10.7, and 0% of organically farmed fields with relative prominence (RP) values of 10.3, 26.5, 0.6, and 0 in the SE, SW, WC, and NW regions, respectively. Across the four regions, other common genera of plant-parasitic nematodes were Helicotylenchus (42.6, RP value, same below), Pratylenchus (26.9), Tylenchorhynchus and related genera (9.4), Xiphinema (5.6), and Paratylenchus (5.3). Aphelenchoides, Meloidogyne, Hoplolaimus, Mesocriconema, and Trichodorus were also detected at low frequencies and/or low population densities. The similarity index of plant-parasitic nematodes between two regions ranged from 0.44 to 0.71 and the similarity increased with decreasing distance between regions. The densities of most plant-parasitic nematodes did not correlate with measured soil factors (organic matter, pH, texture). However, the densities of Pratylenchus correlated negatively with % sand, and Xiphinema was correlated negatively with soil pH.     

Antihelimentic effect of Androctonus crassicauda scorpion venom against Trichuris arvicolae isolated from Psammomys obesus in Egypt

Trichuridae family has a genetic and morphological variability between species affecting rodents, but it is considerably hard to morphologically diagnose species within the genus of Trichuris and the individuals of these species are identified according to their host, as it is known that Trichuris spp. is strictly host-specific. However, some species lack host specificity. So, it is necessary to use molecular data in order to well identify the Trichuris spp. in Egyptian rodents. The host examined in the current research is Psammomys obesus and the molecularly identified species from its cecum is Trichuris arvicolae. In addition, Trichuris arvicolae was subjected to in vitro treatment with Androctonus crassicauda Crude Venom as a model of natural alternative treatment for gastrointestinal nematodes that increasingly develop anthelmintic drug resistance. The changes in Trichuris arvicolae were monitored using scanning electron microscopy, Androctonus crassicauda Crude Venom made a significant ultrastructural surface changes in Trichuris arvicolae, including marked cuticular sloughing, disintegrated bacillary glands, bursting of vulva and edema of anal region. This study was done for closer identification of Trichuris spp. infecting rodents in Egypt and evaluating the efficacy of Androctonus crassicauda Crude Venom in vitro.