Pathogenicity of Pratylenchus penetrans,Heterodera glycines,and Meloidogyne incognita on Soybean Genotypes

The pathogenicity of Heterodera glycines, Meloidogyne incognita, and Pratylenchus penetrans on H. glycines-resistant ''Bryan,'' tolerant-susceptible ''G88-20092,'' and intolerant-susceptible ''Tracy M'' soybean cultivars was tested using plants grown in 800 cm³ of soil in 15-cm-diam. clay pots in three greenhouse experiments. Plants were inoculated with 0, 1,000, 3,000, or 9,000 H. glycines race 3 or M. incognita eggs, or vermiform stages of P. penetrans/pot. Forty days after inoculation, nmnbers of all three nematodes, except H. glycines on Bryan, generally increased with increasing inoculum levels in Experiment I. Heterodera glycines and M. incognita significantly decreased growth only of Tracy M. At 45 and 57 days after inoculation with 6,000 individuals/pot in experiments II and III, respectively, significantly more P. penetrans and M. incognita than H. glycines were found on Bryan. However, H. glycines and M. incognita population densities were greater than P. penetrans on G88-20092 and Tracy M. Growth of Tracy M infected by H. glycines and M. incognita and growth of G88-20092 infected by M. incognita decreased in Experiment III. Pratylenchus penetrans did not affect plant growth. Reduction in plant growth differed according to the particular nematode species and cultivar, indicating that nematodes other than the species for which resistance is targeted can have different effects on cultivars of the same crop species.     

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.     

Separation and Characterization of Heterodera glycines Acetylcholinesterase Molecular Forms

The composition and biochemical properties of acetylcholinesterases isolated from Heterodera glycines were determined. Heterodera glycines contains three separable AChE molecular forms that can be grouped into two classes corresponding to classes A and C found in some other nematode species. The apparent lack of class B AChE is unusual and may have significant behavioral ramifications. The class C enzyme isolated from H. glycines is similar to that from Meloidogyne arenaria and M. incognita but is somewhat more sensitive to AChE inhibitors such as eserine. Heterodera glycines possesses a larger percentage of its total acetylcholinesterase as class C than other nematodes thus far examined.     

Unique Immunogenic Proteins in Heterodera glycines Eggshells

Polyclonal antibodies were raised against Heterodera glycines eggshells to determine the feasibility of developing an immunoassay for H. glycines eggs. An indirect enzyme-linked immunosorbent assay (ELISA) was developed from anfisera collected 10 weeks after the initial injection. From serial dilutions of sonicated eggshells or whole eggs, a sensitivity of detection to 5 ng/ml sonicated eggshells or 1 egg of H. glycines was determined. The method of eggshell preparation had no effect on the antibodies produced; however, the antibodies cross-reacted with sonicated J2 of H. glycines and eggs of Meloidogyne incognita and H. schachtii. Most of the proteins in both life stages of H. glycines and eggs of M. incognita and H. schachtii had similar migration properties when separated on SDS-PAGE gels and stained with Coomassie blue. Western blot analysis, with antisera adsorbed with homogenized J2 of H. glycines, showed proteins that were specifically localized to eggshells of H. glycines. Monoclonal antibodies might provide a useful immunoassay where polyclonal antibodies lack sufficient specificity.     

Ultrastructure of the Head Region of Molting Second-Stage Juveniles of Heterodera glycines with Emphasis on Stylet Formation

The morphology and alterations of infective juvenile (J2) body components with emphasis on the body wall, stomatal wall, stylet, and sensilla of Heterodera glycines were observed. During the molt of J2 to J3, the J2 hypodermis separates from the J2 cuticle and forms an extracellular space, continuous with an invagination of the anterior, center of the J3. The space between the J2 cuticle and the enlarged J3 hypodermal cells is filled with electron-dense material resembling a fluid observed in insects during molt. Regeneration of the J2 during molt was traced in a series of ultrathin sections. The site of stylet regeneration is in the hypodermal and myoepithelial tissues of the invaginated anterior, center of the J3. Four arcade-like cells are related to specific components of the stomatal wall, the stylet cone, and the stylet shaft of the J3. The first and second arcade-like cells are primarily related to stomatal wall development, whereas the third and fourth arcade-like cells are related to stylet cone and shaft development. Spherical, electron-translucent vacuoles that occur in myoepithelial cells just posterior to the arcade-like cells appear to be progenitors of the stylet knobs. Early stages of protractor muscle attachment to the vacuolar membrane were observed.     

Dynamics of Concomitant Populations of Hoplolaimus columbus,Scutellonema brachyurum,and Meloidogyne incognita on Cotton

Cotton seedlings grown in a greenhouse and a growth chamber were inoculated with Scutellonema brachyurum, Hoplolaimus columbus, and Meloidogyne incognita, singly and in all possible combinations, at two initial population (Pi) levels (100 and 300/100 cm³). S. brachyurum alone was not pathogenic to cotton at these population levels. It fed primarily as an ectoparasite but matured and reproduced within the root when it penetrated. Populations of S. brachyurum increased in the presence of H. columbus but were suppressed by M. incognita. H. columbus suppressed dry shoot weights of cotton (P = 0.05) at a Pi of 300/100 cm³ soil. Simultaneous inoculation of H. columbus with either M. incognita or S. brachyurum increased H. columbus populations over treatments with H. columbus alone, both at 60 and 90 d after inoculation. M. incognita suppressed cotton shoot weights significantly (P = 0.05) at both Pi levels. Inoculation with S. brachyurum increased M. incognita populations 60 d after inoculation, while H. columbus suppressed populations of M. incognita. Most larvae of M. incognita did not develop to maturity in the presence of H. columbus. Giant cells aborted and were necrotic 20-25 d after inoculation. Since M. incognita and H. columbus feed on different tissues, the inhibition of M. incognita may have resulted from a physiological effect of H. columbus on the host.     

Ultrastructure of the Anterior Body Region of Marine Nematode Deontostoma californicum

The ultrastructure of the anterior body region of the free-living marine nematode Deontostoma calilornicum was studied by electron microscopy. The body wall consists of a nine-layered cuticle, a cellular hypodermis containing eight nerve bundles, and a well-developed coelomyarian somatic musculature. Nerves in the dorsal, lateral, ventral, and submedian hypodermal chords anterior to the nerve ring were observed with regularity. Structure of subventral somatic setae suggests a mechanoreceptive function. The esophagus is cellular and consists of three marginal cells alternating with an equal number of radial muscle cells, three esophageal glands, and three enteric nerves. The membranes of adjacent esophageal cells are sinuous. Apices of the triradiate lumen are connected with the outer wall of each marginal cell by bands of electron-dense nonmyofibrils, whereas two types of myofilaments run radially between the apophyses of the lumen and the outer walls of radial cells. Each myofibril, which forms hemidesmosomes at both ends, is interpreted to be the morphological equivalent of one sarcomere. Synaptic junctions between the processes of muscles, gland cells, and axons of the enteric nerves are described in detail.     

Monoclonal Antibodies to the Esophageal Glands and Stylet Secretions of Heterodera glycines

Three monodonal antibodies (MAbs) that bound to secretory granules within the subventral esophageal glands of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines, were developed from intrasplenic immunizations of a mouse with homogenates of SCN J2. Two MAbs to the secretory granules within subventral glands and one MAb to granules within the dorsal esophageal gland of SCN J2 were developed by intrasplenic immunizations with J2 stylet secretions. Stylet secretions, produced in vitro by incubating SCN J2 in 5-methoxy DMT oxalate, were solubilized with a high pH buffer and concentrated for use as antigen. Three of the five MAbs specific to the subventral esophageal glands bound to stylet secretions from SCN J2 in immunofluorescence and ELISA assays. Two of these three MAbs also bound to secretory granules within both the dorsal and subventral esophageal glands of young SCN females. All five of the subventral gland MAbs bound to the subventral glands of Heterodera schachtii and one bound to the subventral glands of Globodera tabacum, but none bound to any structures in Meloidogyne incognita or Caenorhabditis elegans.     

Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.     

Glomus fasciculatum,a Weak Pathogen of Heterodera glycines

The occurrence ofchlamydospores of Glomus fasciculatum (Gf) within cysts of the soybean cyst nematode, Heterodera glycines, and the effects of vesicular-arbuscular mycorrhizae on nematode population dynamics and soybean (Glycine max) plant growth were investigated. Chlamydospores occupied 1-24% of cysts recovered from field soil samples. Hyphae of Missouri isolate Gfl penetrated the female nematode cuticle shortly after she ruptured the root epidermis. Convoluted hyphae filled infected eggs, and sporogenesis occurred within infected eggs. G. microcarpum, G. mosseae, and two isolates of Gf were inoculated with H. glycines on plants of ''Essex'' soybeans. Each of the two Gf isolates infected about 1% of the nematode eggs in experimental pot cuhures. The Gfl isolate decreased the number of first-generation adult females 26%, compared with the nonmycorrhizal control. The total numbers of first-generation plus second-generation adult females were similar for both Gf isolates and 29-41% greater than the nonmycorrhizal control. Soybean plants with Gf and H. glycines produced more biomass than did nonmycorrhizal plants with nematodes, but only Gfl delayed leaf senescence.     

Production and Partial Characterization of Stylet Exudate from Adult Females of Meloidogyne incognita

Adult females of Meloidogyne incognita were excised from tomato roots and incubated in 0.04 M phosphate buffered saline, pH 7.4 for 18-72 hours to allow accumulation of stylet exudate. Twenty-four percent of the females produced exudate during the initial 18-hour incubation period; 70% of those females producing exudate initially produced additional exudate during the subsequent 54-hour incubation period. Analysis of exudate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least nine major protein bands. Differential staining with silver and Coomassie Brilliant Blue G-250 stains indicated that three of the bands were glycoproteins. Upon acid hydrolysis, 14 amino acids were detected in the stylet exudate. The basic amino acids lysine, histidine, and arginine comprised 21.8% of the total amino acids detected. No peroxidase activity was detected in the stylet exudates. Data presented extend and generally confirm prior work on the chemical composition of stylet exudate.     

Characteristics and Efficacy of a Sterile Hyphomycete (ARF18), a New Biocontrol Agent for Heterodera glycines and Other Nematodes

A filamentous, nonsporulating fungus, designated Arkansas Fungus 18 (ARF18), was isolated from 9 of 95 populations of Heterodera glycines, the soybean cyst nematode, in Arkansas. In petri dishes, ARF18 parasitized 89% of H. glycines eggs in cysts. The fungus also infected eggs of Meloidogyne incognita and eggs in cysts of Cactodera betulae, H. graminophila, H. lespedezae, H. leuceilyma, H. schachtii, and H. trifolii. In pot tests, reproduction of SCN was 70% less in untreated field soil that was naturally infested by ARF18 than in autoclaved field soil. Although ARF18 grew well at 25 C on cornmeal agar over a wide pH range, it did not sporulate on 28 media and thus could not be identified to genus or species.     

Cuticular Collagenous Proteins of Second-stage Juveniles and Adult Females of Meloidogyne incognita: Isolation and Partial Characterization

Cuticles isolated from second-stage juveniles and adult females of Meloidogyne incognita were purified by treatment with 1% sodium dodecyl sulfate (SDS). The juvenile cuticle was composed of three zones differing in their solubility in β-mercaptoethanol (BME). Proteins in the cortical and median zones were partially soluble in BME, whereas the basal zone was the least soluble. The BME-soluble proteins from the juvenile cuticle were separated into 12 bands by SDS-polyacrylamide gel electrophoresis and characterized as collagenous proteins based on their sensitivity to collagenase and amino acid composition. The adult cuticle consisted of two zones which were dissolved extensively by BME. The basal zone was completely solubilized, leaving behind a network of fibers corresponding to the cortical zone. The BME-soluble proteins from the adult cuticle were separated by electrophoresis into nine bands one of which constituted > 55% of the total BME-soluble proteins. All bands were characterized as collagenous proteins. Collagenous proteins from juvenile cuticles also contained glycoproteins which were absent from the adult cuticles.     

The heterologous expression of a Glycine max homolog of NONEXPRESSOR OF PR1 (NPR1) and α-hydroxynitrile glucosidase suppresses parasitism by the root pathogen Meloidogyne incognita in Gossypium hirsutum

Experiments in Glycine max (soybean) identified the expression of the salicylic acid signaling and defense gene NONEXPRESSOR OF PR1 (NPR1) in root cells (i.e., syncytium) parasitized by the plant parasitic nematode Heterodera glycines undergoing the process of resistance. Gm-NPR1-2 overexpression in G. max effectively suppresses parasitism by H. glycines. The heterologous expression of Gm-NPR1-2 in Gossypium hirsutum impairs the ability of the parasitic nematode Meloidogyne incognita to form root galls, egg sacs, eggs and second-stage juvenile (J2) nematodes. In related experiments, a G. max β-glycosidase (Gm-βg-4) related to Lotus japonicus secreted defense gene α-hydroxynitrile glucosidase LjBGD7 suppresses M. incognita parasitism. The results identify a cumulative negative effect that the transgenes have on M. incognita parasitism and demonstrate that the G. max–H. glycines pathosystem is a useful tool to identify defense genes that function in other agriculturally relevant plant species to plant parasitic nematodes with different strategies of parasitism.     

Optimization of the Heterodera glycines Race Test Procedure

Effects of pot size, length of seedling radicle at the time of inoculation with Heterodera glycines, transplanting after inoculation, type and amount of inoculum, and temperature were tested to determine the optimum procedure for the H. glycines race test. Numbers of H. glycines females extracted from plants in 7.5-cm-d pots tended to be higher than numbers from 10-cm-d pots, but not significantly so. Radicle lengths from 2.5 cm to 7.5 cm had no effect. Transplanting after inoculation reduced the variation in the number of females extracted, but the numbers of females produced were very low. Plump females (40 per pot) or eggs (4,000 per pot) were the best inocula. A constant temperature of 28 C appeared to be optimum. More H. glycines females were extracted from plants 28 days after inoculation than at 35 days. Race tests in which all of these factors were included were still highly variable in the number of H. glycines females extracted from different replications of the same test host. Tests of several susceptible cultivars revealed differences in their capabilities as hosts of H. glycines races.     

Chemical-Mediated Toxicity of N-Viro Soil to Heterodera glycines and Meloidogyne incognita

N-Viro Soil (NVS) is an alkaline-stabilized municipal biosolid that has been shown to lower population densities and reduce egg hatch of Heterodera glycines and other plant-parasitic nematodes; but the mechanism(s) of nematode suppression of this soil amendment are unknown. This study sought to identify NVS-mediated changes in soil chemical properties and their impact upon H. glycines and Meloidogyne incognita mortality. N-Viro Soil was applied to sand in laboratory assays at 0.5%, 1.0%, 2.0%, and 3.0% dry w/w with a nonamended treatment as a control. Nematode mortality and changes in sand-assay chemical properties were determined 24 hours after incubation. Calculated lethal concentration (LC₉₀) values were 1.4% w/w NVS for second-stage juveniles of both nematode species and 2.6 and >3.0% w/w NVS for eggs of M. incognita and H. glycines, respectively. Increasing rates of NVS were strongly correlated (r² = 0.84) with higher sand solution pH levels. Sand solution pH levels and, to a lesser extent, the production of ammonia appeared to be the inorganic chemical-mediated factors responsible for killing plant-parasitic nematodes following amendment with NVS.     

A Revised Classification Scheme for Genetically Diverse Populations of Heterodera glycines

Heterodera glycines, the soybean cyst nematode, is a major yield-limiting pathogen in most soybean production areas worldwide. Field populations of H. glycines exhibit diversity in their ability to develop on resistant soybean cultivars. Since 1970, this diversity has been characterized by a bioassay used to assign a race classification to a population. The value of the race scheme is reflected in the number and quality of resistant soybean cultivars that have been developed and released by soybean breeders and nematologists working in concert. However, the race scheme also has been misapplied as a means of studying H. glycines genotypes, in part due to the use of the term "race." For fungal and bacterial pathogen species, "race" can theoretically be applied to individuals of a population, thus allowing inference of individual genotypes. Application of a race designation to an individual egg or second-stage juvenile (J2) of H. glycines is not possible because a single J2 cannot be tested on multiple hosts. For other nematode species, "race" is defined by host ranges involving different plant species, whereas the H. glycines race test involves a set of lines of the same plant species. Nonetheless, because H. glycines populations vary in genetic diversity, and this variation has implications for management strategies, a mechanism is needed for documenting and discussing population differences. The HG Type scheme described herein avoids the implication of genetic uniformity or predictability in contrast to the way the race scheme has been used.