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.     

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.     

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.     

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 of Cultured Thin Cell Layer Roots of Lycopersicon esculentum by Meloidogyne incognita

A new aseptic culture system for studying interactions between tomato (Lycopersicon esculentum) and Meloidogyne incognita is described. Epidermal thin cell layer explants from peduncles of tomato produced up to 20 adventitious roots per culture in 4-9 days on Murashige &Scoog medium plus kinetin and indole acetic acid. Rooted cultures were transferred to Gamborg''s B-5 medium and inoculated with infective second-stage juveniles. Gall formation was apparent 5 days after inoculation and egg production by mature females occurred within 25 days at 25 C in the susceptible genotypes Rutgers and Red Alert. Resistant genotypes LA655, LA656, and LA1022 exhibited a characteristic hypersensitive response. This system provides large numbers of cultured root tips for studies on the molecular basis of the host-parasite relationship.     

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.     

Effects of Pratylenchus penetrans on the Infection of Strawberry Roots by Gnomonia comari

The fungus Gnomonia comari, causal agent of strawberry leaf blotch, was inoculated at the crown of young axenized strawberry plants growing in sterilized sand. Only the roots were colonized, and the infection was symptomless. When the fungus colonized the roots in the presence of the root lesion nematode Pratylenchus penetrans, the plants were extremely stunted and their root system was necrotic. Fungal conidiospores were found attached to the cuticle of nematodes extracted from soil inoculated with the two pathogens. These findings indicate that P. penetrans could transport conidiospores through soil.     

Population Growth Patterns of Four Species of Aphelenchoides on Fungi

Qualitative and quantitative differences in population growth patterns of Aphelenchoides rutgersi from Florida, A. sacchari from Jamaica, A. dactylocercus from Great Britain, and A. cibolensis from New Mexico were assessed on 28 species of fungi. The patterns of population growth of A. rutgersi and A. sacchari were statistically similar although not identical, and they differed considerably from those of A. dactylocercus and A. cibolensis. It is suggested that A. rutgersi and A. sacchari, from Florida and Jamaica respectively, may be more closely related to each other than to either A. dactylocercus or A. cibolensis.     

Infection of Citrus Roots by Tylenchulus semipenetrans Reduces Root Infection by Phytophthora nicotianae

Bioassays and whole-plant experiments were conducted to investigate the interaction between Tylenchulus semipenetrans and Phytophthora nicotianae. Both organisms are parasites of the citrus fibrous root cortex. Nematode-infected and non-infected root segments were excised from naturally infected field roots and placed on water agar in close proximity to agar plugs of P. nicotianae and then transferred to a Phytophthora-selective medium. At 10 and 12 days, 50% fewer nematode-infected segments were infected by P. nicotianae than non-infected segments. In whole-plant experiments in glass test tubes, sour orange seedlings were inoculated with two densities (8,000 or 80,000 eggs and second-stage juveniles) of T. semipenetrans, and after establishment of infection were inoculated with two densities (9,000 and 90,000 zoospores) of P. nicotianae. In the first experiment, fungal protein was 53% to 65% lower in the roots infected by both organisms than in roots infected by the fungus only. Compared to plants infected only by P. nicotianae, shoot weights were 33% to 50% greater (P ≤ 0.05) in plants infected by both parasites, regardless of inoculum density. Fibrous and tap root weights were 5% to 23% and 19% to 34% greater (P ≤ 0.05), respectively, in nematode-fungus combination treatments compared to the fungus alone. A second experiment was conducted, where plants were infected by the fungus, the nematode, both organisms, or neither organism. The soil mixture pH for 50% of the plants was adjusted from 4.5 to 7.0 to favor nematode infection. A higher rate of nematode infection of plants growing at pH 7.0 compared to pH 4.5 resulted in greater suppression of fungal development and greater inhibition of fungal damage to the plant. Compared to plants infected only by P. nicotianae, shoot and root weights were 37% and 33% greater (P ≤ 0.05), respectively, in plants infected by both parasites. These experiments have revealed antagonism between T. semipenetrans and P. nicotianae in citrus.     

Glucuronidase Expression in Transgenic Tobacco Roots with a Parasponia Promoter on Infection with Meloidogyne javanica

The expression of a g-us reporter gene linked to a Parasponia andersonii hemoglobin promoter has been studied in transgenic tobacco plants after infection by Meloidogyne javanica. Transgenic roots were harvested at different times after nematode inoculation, and stained histochemically for expression of the gus gene. During the early stages of infection (0-2 weeks) there was little expression in giant cells, in contrast to other cells of the root. In later stages of infection (3-6 weeks) there was strong gus expression in giant cells, with virtually no expression in other cells of the root. The Parasponia hemoglobin promoter therefore appears to direct down-regulation of linked genes on induction of giant cells, but up-regulation in mature giant cells. This reflects different metabolic activities in the giant cells depending on their stage of development. The Parasponia hemoglobin promoter may respond to oxygen tension in giant cells. This suggests that oxygen tension may be limited in the metabolically active giant cells that are associated with egg-laying females.     

Infection of Pratylenchus penetrans by Nematode-pathogenic fungi

Eleven fungal isolates were tested in agar dishes for pathogenicity to Pratylenchus penetrans. Of the fungi that produce adhesive conidia, Hirsutella rhossiliensis was a virulent pathogen; Verticillium balanoides, Drechmeria coniospora, and Nematoctonus sp. were weak or nonpathogens. The trapping fungi, Arthrobotrys dactyloides, A. oligospora, Monacrosporium dlipsosporum, and M. cionopagum, killed most of the P. penetrans adults and juveniles added to the fungus cultures. An isolate of Nematoctonus that forms adhesive knobs trapped only a small proportion of the nematodes. In 17-cm³ vials, soil moisture influenced survival of P. penetrans in the presence of H. rhossiliensis; nematode survival decreased with diminishing soil moisture. Hirsutella rhossiliensis and M. ellipsosporum were equally effective in reducing numbers of P. penetrans by 24-25% after 4 days in sand. After 25 days in soil artificially infested with H. rhossiliensis, numbers of P. penetrans were reduced by 28-53%.     

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.     

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.     

Effect of Hirsutella rhossiliensis on Infection of Potato by Pratylenchus penetrans

We evaluated the ability of the nematode-pathogenic fungus Hirsutella rhossiliensis (Deuteromycotina: Hyphomycetes) to reduce root penetration and population increase of Pratylenchus penetrans on potato. Experiments were conducted at 24 C in a growth chamber. When nematodes were placed on the soil surface 8 cm from a 14-day-old potato cutting, the fungus decreased the number entering roots by 25%. To determine the effect of the fungus on population increase after the nematodes entered roots, we transplanted potato cuttings infected with P. penetrans into Hirsutella-infested and uninfested soil. After 60 days, the total number of nematodes (roots and soil) was 20 ± 4% lower in Hirsutella-infested than in uninfested soil.     

Penetration of Celery and Alfalfa Roots by Pratylenchus penetrans as Affected by Temperature

A greater percentage of females than juveniles or males of P. penetrans penetrated celery roots grown in infested soil at 5, 18, or 30 C; the difference was greatest at 5 C. The time of initial penetration of alfalfa seedlings inoculated with single nematodes on water agar varied with temperature. Females penetrated the seedlings earlier and over a wider range of temperatures than did males or juveniles. The rate of penetration was highest for females. After initial penetration, the penetration rate decreased with time. At 13-28 C, approximately 80% of roots were penetrated by females and only 25-30% by males and juveniles by the end of the experiment.     

Predisposition of Broadleaf Tobacco to Fusarium Wilt by Early Infection with Globodera tabacum tabacum or Meloidogyne hapla

In greenhouse experiments, broadleaf tobacco plants were inoculated with tobacco cyst (Globodera tabacum tabacum) or root-knot (Meloidogyne hapla) nematodes 3, 2, or 1 week before or at the same time as Fusarium oxysporum. Plants infected with nematodes prior to fungal inoculation had greater Fusarium wilt incidence and severity than those simultaneously inoculated. G. t. tabacum increased wilt incidence and severity more than did M. hapla. Mechanical root wounding within 1 week of F. oxysporum inoculation increased wilt severity. In field experiments, early-season G. t. tabacum control by preplant soil application of oxamyl indirectly limited the incidence and severity of wilt. Wilt incidence was 48%, 23%, and 8% in 1989 and 64%, 60%, and 19% in 1990 for 0.0, 2.2, and 6.7 kg oxamyl/ha, respectively. Early infection of tobacco by G. t. tabacum predisposed broadleaf tobacco to wilt by F. oxysporum.     

Competition between Heterodera glycines and Meloidogyne incognita or Pratylenchus penetrans: Independent Infection Rate Measurements

Competition on soybean between Heterodera glycines (race 3) and Meloidogyne incognita or H. glycines and Pratylenchus penetrans were investigated in greenhouse experiments. Each pair of nematode species was mixed in 3-ml suspensions at ratios of 1,000:0, 750:250, 500:500, 250:750, and 0:1,000 second-stage juveniles or mixed stages for P. penetrans. Nematodes from a whole root system were counted and infection rates standardized per 1,000 nematodes (per replication) prior to testing the null hypothesis through a lack-of-fit F-test. Although the effect of increasing H. glycines proportions on the infection rate of M. incognita was generally adverse, the rate deviated significantly from a trend of linear decline at the 75% H. glycines level in one of two experiments. All lack-of-fit F-tests for the H. glycines and P. penetrans mix were significant, indicating that infection rates for both nematodes varied considerably across inocula. The infection rate of H. glycines decreased with increasing P. penetrans proportions. The rate of P. penetrans infection increased with increasing H. glycines proportions up to the 50% level, but declined at the 75% level. Competition had no effect on nematode development. The general adverse relationships between M. incognita and H. glycines and those between P. penetrans and H. glycines showed a linear trend. The relationship between H. glycines and P. penetrans indicates that the former may be competitive when present at higher proportions than the latter. In this study we have evaluated nematode competition under controlled conditions and provide results that can form a basis for understanding the physical and physiological trends of multiple nematode interactions. Methods critical to data analyses also are outlined.     

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.     

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.