Modification of the Oligidic Medium for Axenic Culture of Aphelenchoides rutgersi

Aphelenchoides rutgersi was axenically cultured in modified Soytone, yeast extract, lyophilized chick embryo extract medium (3% ST:2% YE:20% CEE-L, w/v:w/v:v/v). Earlier formulations used 10% CEE, v/v, before the manufacturer changed the preparation. After reestablishing A. rutgersi in medium that permitted continuous subcultivadon and reproduction, a second medium was tested that contained 0.5% sucrose and 0.5% Lipid Concentrate. The commercially available Lipid Concentrate made it possible to incorporate nonaqueous soluble chemicals into the medium. In addition, 0.1% Fast Green #3 was added to both media to visually demonstrate active ingestion of nutriment.     

Infection of Narcissus Roots by Aphelenchoides subtenuis

The widespread destruction of commercially grown bulbs of Narcissus tazetta papyraceus (Paper White) has been reported in Israel. This phenomenon is usually characterized by a premature yellowing of the foliage, accompanied by root rot and dark, sunken basal plates. This study confirmed thatAphelenchoides subtenuis is the main cause of the basal plate disease of Narcissus. In contrast to other Aphelenchoides species, which feed on stems or leaves, A. subtenuis penetrates Narcissus roots. In our experiments, in winter (6 to 8 weeks after penetration), nematodes laid their eggs in the root parenchymal cells without inducing obvious symptoms on foliage or roots. Toward spring, juveniles became numerous throughout the parenchymal cells of the root cortex. Consequently, the root system collapsed rapidly, at the usual peak of bulb and foliage production. Bulbs of infected plants were small and weighed less than those of uninfected plants, and foliage became necrotic prematurely. At that time, in field conditions, secondary elements like Fusarium penetrate the bulb and cause it to rot, given this syndrome the common name of basal plate disease. To our knowledge, this is the first report of an Aphelenchoides species as a root pathogen.     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.     

Influence of Temperature on Population Development of Eight Species of Pratylenchus on Soybean

In a soil temperature study, population increase on ''Clark 63'' soybeatt was most rapid at 30 C in Pratylenchus alleni, P. brachyurus, P. cofleae, P. neglectus, P. scribneri, and P. zeae and at 25 C in P. penetrans and P. vulnus. The last two were the only species that reproduced at 15 C. Populations of all species increased over the range of 20-30 C, except those of P. neglectus at 20 C and P. coffeae, which was not tested below 25 C. Only P. brachyurus, P. neglectus, P. scribneri and P. zeae reproduced at 35 C. At their optimum temperatures, P. scribneri exhibited the greatest population increase, 1248-fold, and P. penetrans the least, 32-fold. This is the first report of soybean as a host for P. vulnus.     

Taxonomic Notes on Four Species of Panagrellus Thorne (Nematoda: Cephalobidae)

Four populations of Panagrellus redivivus (Linnaeus, 1767) Goodey, 1945, and syntypes of P. dubius Sanwal, 1960, P. redivivoides (Goodey, 1943) Goodey, 1945, P. pycnus Thorne, 1938, P. zymosiphilus (Brunold, 1950) Brunold, 1954, and P. leucocephalus Steiner, 1936 were examined. Additional information on the morphology of spicules and stoma is presented. P. zymosiphilus is synonymized with P. redivivoides and Rühm''s synonymization of P. silusiae and P. leucocephalus with P. redivivus is supported. Lectolypes for P. pycnus and P. redivivoides are designated.     

Observations on the Foliar Nematode,Aphelenchoides besseyi,Infecting Tuberose and Rice in India

The foliar nematode Aphelenchoides besseyi causes white tip disease in rice (Oryza sativa L.) and floral malady in tuberose (Polianthes tuberosa L.). This nematode is widely distributed in the rice fields of many states of India, including West Bengal (WB), Andhra Pradesh (AP), Madhya Pradesh (MP) and Gujarat (GT). In order to generate information on intraspecific variations of A. besseyi as well as to confirm the identity of the nematode species infecting these important crops, morphological observation was undertaken of A. besseyi isolated from tuberose and rice from WB and rice from AP, MP and GT. The molecular study was only done for rice and tuberose populations from AP and WB. The variations were observed among the populations in the tail, esophageal and anterior regions, including the occurrence of four as well as six lateral lines in the lateral fields. The morphometrics of observed populations showed variations and those could be regarded as a consequence of host-induced or geographical variations. PCR amplification of the rDNA ITS 1 and 2 region of rice (AP) and tuberose (WB) populations of A. besseyi generated one fragment of approximately 830 bp, and the size of the ITS region was 788 bp and 791 bp for tuberose and rice population, respectively. Alignment of the two sequences showed almost 100% similarity. Blast analysis revealed a very high level of similarity of both the Indian strains to a Russian population. The Indian and Russian strains could be differentiated using restriction enzyme Bccl. Host tests revealed that rice (cv. IET 4094), oat (cv. OS-6) and teosinte (cv. TL-1) showed a typical distortion due to the infection of A. besseyi. Five germplasm lines of oat showed no infection of the nematode under field conditions. Local cultivars of onion, maize, chrysanthemum, gladiolus, and Sorghum halepense were also not infected by A. besseyi.     

Ruscus hypophyllum: A New Host for Aphelenchoides fragariae

Aphelenchoides fragariae was isolated from the phylloclades of the ornamental plant Ruscus hypophyllum (Liliaceae). Rotylenchus buxophilus, Scutellonema brachyurum, and Meloidogyne were identified as the most common plant-parasitic nematodes in the soil near the roots. The pathology and life history of A. fragariae were closely related to the climate. To our knowledge, this is the first report of R. hypophyllum as a host of plant-parasitic nematodes.     

Infection Behavior and Overwintering Survival of Foliar Nematodes, Aphelenchoides fragariae, on Hosta

We studied the pathogenicity and overwintering survival of the foliar nematode, Aphelenchoides fragariae, infecting Hosta spp. Nematodes applied to either lower or upper sides of noninjured and injured hosta leaves were able to infect and produce typical symptoms on nine cultivars. Leaves of only four cultivars (Borschi, Fragrant Blue, Patomic Pride, and Olive Bailey Langdon) showed no symptoms of nematode infection. The nematodes overwintered as juveniles and adults in soil, dry leaves, and dormant buds, but not in roots. Nematode winter survival was higher in dormant buds and soil from the polyhouse than in an open home garden. Of the nematodes found in the dormant buds, 35% to 79% were located between the first two outside layers of the buds. The nematodes tolerated 8 hr exposure to 40°C and −80°C in leaf tissues. Relative humidity influenced nematode migration from soil to leaves. The presence of nematodes only on the outer surface of foliage (leaves and petioles) confirmed the migration of A. fragariae on the surface of the plants. Of the total number of nematodes found on the foliage, 25% to 46% and 66% to 77% were alive at 90% and 100% relative humidity, respectively, suggesting that high moisture is required for the survival and upward movement of nematodes. We conclude that A. fragariae can overwinter in soil, infected dry leaves, and dormant buds and migrate in films of water on the outer surface of the plant during spring to leaves to initiate infection.     

Species of Phytophthora and Pythium as Nematode-destroying Fungi

Pythium monospermum,, P. aphanidermatum, and Phytophthora palmivora were found to be capable of destroying certain nonstylet-bearing nematodes through endozoic parasitism by hyphae from ingested zoospores. Hyphae of P. monospermum parasitized nematode eggs but could not capture or otherwise prey upon living nematodes. We suggest that endoparasitism of free-living nematodes may be common among Oomycetes in nature.     

Effectiveness of a Hot Water Drench for the Control of Foliar Nematodes Aphelenchoides fragariae in Floriculture

Effectiveness of a hot water drench for the control of Aphelenchoides fragariae infesting hosta (Hosta sp.) and ferns (Matteuccia pensylvanica) was studied. Drenching with hot water at 70 °C and 90 °C in October reduced (P < 0.05) A. fragariae in the soil but not in the leaves relative to the control (25 °C) 300 days after treatment (DAT). Plants drenched with 90 °C water had lower numbers of nematode-infected leaves per plant than those treated with 25 °C and 70 °C water (P < 0.05). Hot water treatments had no adverse effect on the growth parameters of hosta. Boiling water (100 °C) applied once a month for 3 consecutive months (April, May, June) consistently reduced the number of infected leaves and the severity of infection relative to the control 150 DAT in hosta but not in ferns (P < 0.05). Boiling water (100 °C) caused a 67% reduction in A. fragariae population in hosta leaves, 50% in fern fronds, and 61% to 98% in the soil over the control 150 DAT. A boiling water drench had no effect on the fern growth but caused 49% and 22% reduction in the number and size of hosta leaves, respectively, over the control in 2002. We conclude that 90 °C water soil drench in the autumn or early spring could prove effective in managing foliar nematodes on hosta in nurseries and landscapes.     

Aphelenchoides microstylus n. sp. and Seinura onondagensis n. sp. (Nemata: Aphelenchina) from New York

Aphelenchoides microstylus n. sp. and Seinura onondagensis n. sp., a nematode predator, are described from dead Scots pine (Pinus sylvestris L.) in Onondaga County, New York. Females of A. microstylus are 370 to 485 µm long. The body is slender and tapers posteriorly to an amucronate, pointed terminus. The head is continuous with the body, and lips bear a stylet guide. Diagnostic characters of females are three incisures in the lateral field, a short stylet (6-7.5 µm) with small basal knobs, a single row of oocytes, and a long postuterine sac (25-50 µm). Males are characterized by small spicules (10-11µm); two pairs of post-anal, subventral papillae; and a single row of spermatocytes. A bursa and gubernaculum are absent. Seinura onondagensis females are characterized by a body of moderate length (475-595 µm), finely annulated cuticle, and a slightly set-off head. Diagnostic characters are four incisures in the lateral field, long stylet without basal knobs (17-22 µm), single row of oocytes, and presence of a postuterine sac (14-38 µm). Males are unknown. The monospecific genus Indaphelenchus is proposed as a synonym of Seinura, and S. siddiqii n. comb. is proposed for the only species, I. siddiqii.     

Relative Susceptibility of Four Pine Species to Infection by Pinewood Nematode

Mature trees of eastern white, jack, Scotch, and shortleaf pines were inoculated with 25,000-34,000 pinewood nematodes, Bursaphelenchus xylophilus, isolated from infected Scotch pines in Missouri. Equal numbers of trees of each species inoculated with distilled water served as controls. Nine of fifteen Scotch pines died within 4 months of nematode infection or during the winter and early spring following infection. A single eastern white and shortleaf pine died. No jack pines died. A single Scotch pine control died, apparently the result of natural nematode infection. No other controls died. Mean oleoresin flow did not differ among nematode-inoculated jack and shortleaf pines and their respective controls. Oleoresin flow in nematode-inoculated eastern white and Scotch pines was significantly lower than in their controls. Oleoresin flow was temporarily reduced in mortality-resistant eastern white and Scotch pines following nematode infection. Thus a sublethal impact of nematode infection on mortality-resistant host trees was documented.     

Greenhouse Studies on the Effect of Marigolds (Tagetes spp.) on Four Meloidogyne Species

The effects of preplanted marigold on tomato root galling and multiplication of Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla were studied. Marigold cultivars of Tagetes patula, T. erecta, T. signata, and a Tagetes hybrid all reduced galling and numbers of second-stage juveniles in subsequent tomato compared to the tomato-tomato control. All four Meloidogyne spp. reproduced on T. signata ''Tangerine Gem''. Several cultivars of T. patula and T. erecta suppressed galling and reproduction of Meloidogyne spp. on tomato to levels lower than or comparable to a fallow control. Phytotoxic effects of marigold on tomato were not observed. Several of the tested marigold cultivars are ready for full-scale field evaluation against Meloidogyne spp.     

Systematics of the Monodelphic Species of Trichodoridae (Nematoda: Diphtherophorina) With Descriptions of a New Genus and Four New Species

To show the relationship of the monodelphic species of Trichodoridae with the nominal taxa, the genus Monotrichodorus is redefined, with the addition of one new species. The new genus Allotrichodorus is proposed on the basis of new species found in Brazil; and one new species is described in the genus Paratrichodorus.     

Seasonal Fluctuations of Soil and Tissue Populations of Ditylenchus dipsaci and Aphelenchoides ritzemabosi in Alfalfa

Population dynamics of A. ritzemabosi and D. dipsaci were studied in two alfalfa fields in Wyoming. Symptomatic stem-bud tissue and root-zone soil from alfalfa plants exhibiting symptoms of D. dipsaci infection were collected at intervals of 3 to 4 weeks. Both nematodes were extracted from stem tissue with the Baermann funnel method and from soil with the sieving and Baermann funnel method. Soil moisture and soil temperature at 5 cm accounted for 64.8% and 61.0%, respectively, of the variability in numbers of both nematodes in soil at the Big Horn field. Also at the Big Horn field, A. ritzemabosi was found in soil on only three of the 14 collection dates, whereas D. dipsaci was found in soil on 12 dates. Aphelenchoides ritzemabosi was found in stem tissue samples on 9 of the 14 sampling dates whereas D. dipsaci was found on all dates. Populations of both nematodes in stem tissue peaked in October, and soil populations of both peaked in January, when soil moisture was greatest. Numbers of D. dipsaci in stem tissue were related to mean air temperature 3 weeks prior to tissue collection, while none of the climatic factors measured were associated with numbers of A. ritzemabosi. At the Dayton field, soil moisture plus soil temperature at 5 cm accounted for 98.2% and 91.4% of the variability in the soil populations of A. ritzemabosi and D. dipsaci, respectively. Aphelenchoides ritzemabosi was extracted from soil at two of the five collection dates, compared to extraction of D. dipsaci at three dates. Aphelenchoides ritzemabosi was collected from stem tissue at six of the seven sampling dates while D. dipsaci was found at all sampling dates. The only environmental factor that was associated with an increase in the numbers of both nematodes in alfalfa stem tissue was total precipitation 1 week prior to sampling, and this occurred only at the Dayton field. Numbers of A. ritzemabosi in stem tissue appeared to be not affected by any of the environmental factors studied, while numbers of D. dipsaci in stem tissue were associated with cumulative monthly precipitation, snow cover at time of sampling, and the mean weekly temperature 3 weeks prior to sampling. Harvesting alfalfa reduced the numbers of A. ritzemabosi at the Big Horn field and both nematodes at the Dayton field.     

Studies on nutrient media for plant parasitic nematodes. IV. Physical aspects of culturing Aphelenchoides rutgersi

Little attention is usually given to the physical effects media have upon nematodes even though physical parameters such as pH, temperature, viscosity, osmotic pressure, waste accumulation, and other factors might obscure the positive value of modifications of the nutriment. It was calculated by extrapolation from experimental evidence that the numbers of A. rutgersi would not increase above 19 atm osmotic pressure, 52 cP viscosity, or a specific gravity exceeding 1.4. Osmotic pressure had the greatest effect on reproduction of A. rutgersi. Optimum temperature for reproduction of A. rutgersi was 23 C in M-6 and pH 7.0 was optimal for the nutrient-environment. Similarly, mobility was not modified by adjusting osmotic pressure, viscosity, or specific gravity within the above limits. It was estimated that for obtaining sufficient oxygen the radius of fluid surrounding a nematode should not exceed a maximum of 1 mm, yet reproduction was unaffected in fluids up to 9 mm deep.     

Effect of Arbuscular Mycorrhizal Inoculations on Seedling Growth and Biomass Productivity of Two Bamboo Species

A study was conducted to identify suitable arbuscular mycorrhizal (AM) fungi for inoculation of Bambusa bambos and Dendrocalamus strictus at nursery stage for increasing growth and productivity. Twelve AM species, isolated from bamboo and other common trees of Bundelkhand were used for inoculations. In B. bambos, total dry weight and phosphorus (P) uptake were significantly increased by all studied fungi and shoot length was increased by eight AM inoculants. Maximum mycorrhizal dependency (MD) was recorded for Acaulospora scrobiculata (44.2%), followed by Glomus cerebriforme (41.6%) and G. intraradix (41.0%). In D. strictus, all tested AM inoculants significantly increased shoot length, dry shoot weight and P uptake, except Glomus 1. Dry root weight was significantly increased by only two inoculants namely, G. cerebriforme and G. etunicatum. Total dry weight was significantly increased by eight AM fungi. Maximum MD was recorded for G. cerebriforme (62.9%), followed by G. diaphanum (55.0%) and G. etunicatum (51.3%). Thus, the results showed that utilization of effective AM fungi can enhance the productivity of bamboo in the region.     

An Aphelenchoides sp. nematode Parasitic of Polianthes tuberosa in the Mekong Delta

Polianthes tuberosa is a commercially valuable flower crop in the Mekong Delta of Vietnam that is propagated by the harvesting and planting of bulbs. The cultivation of P. tuberosa is complicated by an endemic nematode infection that damages a high proportion of the plants. Based on morphological criteria and ribosomal RNA gene sequencing, we have determined that the infection is caused by an Aphelenchoides sp. nematode and is most likely Aphelenchoides besseyi. By scoring various parts of harvested plants with flowers for the presence of viable nematodes over a period of six months, we determined that the nematode is an ectoparasite that can survive the intercrop periods, especially in the bulbs that are used to plant new crops. A comparison of farming practices in the Mekong Delta failed to identify useful control methods, but rather indicated that fields that have cultivated P. tuberosa for the longest periods suffer the worst damage from the nematode infection. Finally, we demonstrated that the nematode is capable of infecting 30 rice cultivars but does not cause the white tip disease usually associated with A. besseyi infection.