Extraction of Root-associated Meloidogyne incognita and Rotylenchulus reniformis

A technique based on physical maceration of root tissue was developed to extract vermiform and swollen stages of Meloidogyne incognita and Rotylenchulus reniformis. Experiments conducted on soybean and tomato evaluated the efficiency of method (stir, grind), NaOC1 concentration (0%, 0.5%), and duration (lx, 2x) on extraction of nematodes and eggs from 60-day-old populations. Root-associated populations of R. reniformis were considerably lower than those of M. incognita, so development of the method focused on the latter. Grinding liberated more nematodes than stirring, but the reverse was true for egg extraction. Among grinding treatments, a duration of 10 seconds in 0.5% NaOCl provided the most efficient extraction of nematodes and eggs. Among stirring treatments, a duration of 10 minutes in 0.5% NaOCl provided the most efficient extraction of eggs. These techniques were compared on soybean roots 30 days older than those on which the procedures were first evaluated, with consistent results.     

Reproduction of Pratylenchus penetrans on Potato and Crops Grown in Rotation with Potato

The relative suitability of potato and crops frequently grown in rotation with potato as hosts for Pratylenchus penetrans was evaluated. Suitability of rye, wheat, corn, oat, sorgho-sudangrass, and potato were compared in pot studies based on ratios of final population : initial population density and densities of nematodes in roots at harvest. Population densities increased more on potato, oat, and corn than on rye, wheat, and sorgho-sudangrass. There were no differences among the four rye cultivars or between the two oat cultivars in host suitability. Population increases were not related to root weight or consistently to nematode densities in roots. Although rye and wheat were equally suitable hosts in pot studies, P. penetrans increased more on wheat than on rye in a field study, indicating that reproduction was reduced or mortality was increased on rye under field conditions.     

Sterile Culture of Rotylenchulus reniformis on Tomato Root with Gellan Gum as a Supporting Medium

Rotylenchulus reniformis was repeatedly propagated in sterile excised tomato roots growing on modified White''s medium with gellan gum as the support. Gellan gum provided an optically clear support medium that could be liquified by adding 5 mM disodium ethylenediaminetetraacetate (EDTA) to facilitate nematode extraction. Liquefaction of the gellan-gum medium by EDTA allowed efficient recovery of eggs and vermiform stages of R. reniformis. Extraction efficiency was quantified with Radopholus similis as a test organism. The efficiency of extracting R. similis from the gellan gum did not vary with the concentrations of EDTA tested.     

Optimal Levels of Meloidogyne incognita Inoculum for Infection of Tomato and Peach in Vitro

Penetration of second-stage juveniles (J2) of Meloidogyne incognita into tomato root explants and in vitro propagated peach plantlet roots were compared. Five inoculum levels were used: 25, 50, 75, 100, and 200 J2 for tomato; and 50, 100, 200, 500, and 1,000J2 for peach. The greatest root penetration into tomato was 30% at the 75 J2 level, but the maximum penetration into peach roots was only 8% at the 200 J2 level. The difference (P = 0.05) in penetration of M. incognita at all inoculum levels into these two hosts indicates that penetration versus inoculum density for in vitro studies need to be determined for different plant species.     

Early Growth of Soybean as Altered by Heterodera glycines,Phenamiphos and/or Alachlor

Greenhouse and field experiments were conducted to determine the effects of phenamiphos and/or alachlor on early growth of soybean, root morphology, and infection and resurgence of Heterodera glycines (race 1). All tests were planted to ''Ransom'' soybeans. In greenhouse experiments without nematodes, root growth was inhibited at 5 days by alachlor treatments and at 10 days by phenamiphos treatments; with nematodes, phenamiphos treatments enhanced root growth. Phenamiphos also suppressed early penetration of soybean roots by H. glycines in the greenhouse. Early soybean growth parameters among treatments were generally similar in the field. Nematode penetration was limited with treatments containing phenamiphos at one location. Plants treated with only alachlor had less nematode infection than did the control; however, plants treated with herbicide/nematicide combinations had more nematode penetration than did plants treated with phenamiphos alone. Alterations of root growth and interference with the efficacy of phenamiphos are two processes by which alachlor may enhance soybean susceptibility or suitability to H. glycines.     

Variability among Populations of Meloidogyne arenaria

Variability in reproduction and pathogenicity of 12 populations of Meloidogyne arenaria race 1 was evaluated on Florunner peanut, Centennial soybean, Rutgers tomato, G70, K326, and Mc944 tobacco, and Carolina Cayenne, Mississippi Nemaheart, and Santanka pepper. Differences among M. arenaria populations in rates of egg production 45 days after inoculation were observed for all cultivars except Santanka pepper. Differences among populations in dry top weights or fresh root weights were recorded on all cultivars. Numbers of nematode eggs produced on Florunner peanut varied from 3,419 to 11,593/g fresh root weight. On resistant tobacco cultivars (G70 and K326), one nematode population produced high numbers of eggs (12,042 and 6,499/g fresh root weight on G70 and K326, respectively), whereas the other populations produced low numbers of eggs (less than 500 eggs/g fresh root weight on both cultivars). Two variant M. arenaria race 1 populations were identified by factor analysis of reproductive rates on all nine cultivars. Differences m reproduction and pathogenicity observed among populations would affect the design of sustainable management systems for M. arenaria.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.     

Reproduction and Development of Meloidogyne incognita and M. javanica on Guardian Peach Rootstock

Guardian peach rootstock was evaluated for susceptibility to Meloidogyne incognita race 3 (Georgia-peach isolate) and M. javanica in the greenhouse. Both commercial Guardian seed sources produced plants that were poor hosts of M. incognita and M. javanica. Reproduction as measured by number of egg masses and eggs per plant, eggs per egg mass, and eggs per gram of root were a better measure of host resistance than number of root galls per plant. Penetration, development, and reproduction of M. incognita in Guardian (resistant) and Lovell (susceptible) peach were also studied in the greenhouse. Differences in susceptibility were not attributed to differential penetration by the infectivestage juveniles (J2) or the number of root galls per plant. Results indicated that M. incognita J2 penetrated Guardian roots and formed galls, but that the majority of the nematodes failed to mature and reproduce.     

Effects of Etomopathiogenic Nematodes on Meloidogyne javanica on Tomatoes and Soybeans

Two Hawaiian isolates of Steinernema feltiae MG-14 and Heterohabditis indica MG-13, a French isolate of S. feltiae SN, and a Texan isolate of S. riobrave TX were tested for their efficacy against the root-knot nematode, Meloidogyne javanica, in the laboratory and greenhouse. Experiments were conducted to investigate the effects of treatment application time and dose on M. javanica penetration in soybean, and egg production and plant development in tomato. Two experiments conducted to assess the effects of entomopathogenic nematode application time on M. javanica penetration demonstrated that a single application of 10⁴ S. feltiae MG-14 or SN infective juveniles per 100 cm³ of sterile soil, together with 500 (MG-14) or 1,500 (SN) second-stage juveniles of M. javanica, reduced root penetration 3 days after M. javanica inoculation compared to that of a water treatment. Entomopathogenic nematode infective juveniles applied to assess the effects on M. javanica egg production did not demonstrate a significant reduction compared to that of the water control treatment. There was no dose response effect by Steinernema spp. On M. javanica root penetration or egg production. Steinernema spp. did not affect the growth or development of M. javanica-infected plants, but H. indica MG-13-treated plants had lower biomass than untreated plants infected with M. javanica. Infective juveniles of S. riobrave TX, S. feltiae SN, and MG-14 but not those of H. indica MG-13 were found inside root cortical tissues of M. javanica-infected plants. Entomopathogenic nematode antagonism to M. javanica on soybean or tomato was insufficient in the present study to provide a consistent level of nematode suppression at the concentrations of infective juveniles applied.     

Population Dynamics of Heterodera glycines in Conventional Tillage and No-Tillage Soybean/Corn Cropping Systems

The effects of no-tillage (NT), conventional tillage (CT), and crop rotation on soybean yield and population dynamics of Heterodera glycines were compared during a 7-year study in a silty clay loam soil with 6% organic matter. Either H. glycines-resistant ''Linford'' soybean or susceptible ''Williams 82'' soybean was rotated with corn and grown on 76-cm-wide rows in both tillage systems. Soybean was planted in 1994, 1996, 1998, 1999, and 2000. Yield of Linford was significantly greater than Williams 82 in all years. Soybean yield was affected by tillage in 1999 and 2000. No-tillage production tended to support more reproduction (R = number of eggs at harvest/number of eggs at planting) on both cultivars. The largest R for Williams 82 were in 1998: 58.35 for NT plots and 11.78 for CT plots. For Linford, the largest R were 12.09 for NT plots in 1996, and 3.71 for CT in 1999. When corn was planted, R decreased more in NT. When soybean was planted in years subsequent to 1994, numbers of eggs at harvest (Pf) were greater for Williams 82 NT than for Williams 82 CT or Linford in both tillage systems; however, crop rotation with corn negated these population increases. The soil became suppressive to H. glycines in 1999 and was suppressive in 2000. After the 3 years of continuous soybean, Pf per 250 cm[sup3] soil were 2,870 for Williams 82 NT, 791 for Williams 82 CT, 544 for Linford NT, and 990 for Linford CT in 2000, compared with Pf of 13,100 for Williams 82 NT, 15,000 for Williams CT, 2,360 for Linford NT, and 2,050 for Linford CT in 1994. Describing population dynamics solely on the basis of R was not adequate, but also required independent examination of initial populations following overwintering and Pf after the growing season. Planting soybean either NT or CT in rotation with corn did not result in long-term increases in numbers of H. glycines eggs.     

Ethylene Production by Meloidogyne spp.-Infected Plants

Gall size and rates of ethylene production by various hosts infected with Meloidogyne javanica and by excised tomato root cultures infected with M. javanica or M. hapla were measured. Infection with M. javanica increased the rate of ethylene production in dicotyledonous plants (cabbage, pea, carrot, cucumber, carnation, and tomato), but not in infected monocotyledonous plants (corn, wheat, and onion). Nematode infection induced large galls on roots of dicotyledonous, but not monocotyledonous, plants. Excised tomato roots in culture infected with M. javanica produced ethylene at high rates and formed large galls, whereas roots infected with M. hapla produced ethylene at low rates and induced smaller galls.     

Penetration of Crotalaria juncea,Dolichos lablab,and Sesamum indicum Roots by Meloidogyne javanica

Penetration of Crotalaria juncea (PI 207657 and cv. Tropic Sun) Dolichos lablab cv. Highworth, and Sesamum indicum by juveniles (J2) of Meloidogyne javanica was assessed to investigate the mechanism by which these plants may reduce nematode numbers in the field. Growth chamber experiments were conducted at 25 C, with vials containing 90 g sand infested with 450 J2; tomato (UC 204 C) was included as a susceptible host. Fifteen days after inoculation, roots were stained and the nematodes within stained roots were counted. Both C. juncea lines were highly resistant to penetration, as they contained significantly fewer nematodes per cm of root and per root system than the other plants. Although containing more nematodes per cm of root than C. juncea, S. indicum and D. lablab had significantly fewer nematodes per root system and per cm of root than tomato. Roots were significantly longer in the plants with the lowest nematode penetration. Although C. juncea, D. lablab, and S. indicum may have potential utility as cover or rotation crops in soil infested with M. javanica, further quantitative information on the reproduction of M. javanica and other nematodes in these plants is needed.     

Meloidogyne incognita Inoculum Source Affects Host Suitability and Growth of Yellow Nutsedge and Chile Pepper

Meloidogyne incognita (Mi) reproduction and host plant responses in chile pepper (Capsicum annuum) and yellow nutsedge (Cyperus esculentus = YNS) to three sources of inoculum obtained by rearing a single Mi population on chile, YNS, and tomato were evaluated in two factorial greenhouse experiments. The interactive effects of Mi inoculum source and crop-weed competition were determined. In the absence of YNS competition, chile growth was reduced less by Mi inoculum from chile than by inoculum from YNS or tomato. When YNS was present, chile root weight was not affected and shoot weight increased with Mi initial inoculation, regardless of inoculum source. Chile plants inoculated with Mi from tomato exhibited double the nematode reproduction observed with inoculum from chile or YNS. With chile present, Mi reproduction on YNS was nearly three times greater with inoculum from tomato, but reproduction was similar among inoculum sources when chile was absent. Reductions in YNS root mass due to competition from chile failed to reduce the total number of Mi eggs produced on YNS plants. Differences in total Mi reproduction among inoculum sources were not attributable to differences in root growth or plant competition. This study illustrates the influence of Mi-YNS interactions and previous hosts on severity of Mi infection.     

Crop Rotation and Races of Meloidogyne incognita in Cotton Root-knot Management

The influence o f various crop rotations and nematode inoculum levels on subsequent population densities of Meloidogyne incognita races 1 and 3 were studied in microplots. Ten different 3-year sequences o f cotton, corn, peanut, or soybean, all with cotton as the 3rd-year crop, were grown in microplots infested with each race. Cotton monoculture, two seasons o f corn, or cotton followed by corn resulted in high race 3 population densities and severe root galling on cotton the 3rd year. Peanut for 2 years preceding cotton most effectively decreased the race 3 population and root galls on cotton the 3rd year. Race 1 did not significantly influence cotton growth or yield at initial populations of up to 5,000 eggs/500 cm³ soil. At 5,000 eggs/500 cm³, cotton growth was suppressed by race 3 but yield was not affected.     

Distribution of Pratylenchus scribneri between Root and Soil Habitats

The abundance of Pratylenchus scribneri in soil and root habitats was compared in potato and corn plots during 1986-88. Nematodes were extracted from 100-cm³ soil samples and the roots contained within the samples. The percentage of the population recovered from soil, similar among years and crops, averaged ca. 50% at the beginning and end of the growing season and ca. 20% from early to late season. Proportionately more adults and fourth-stage juveniles than younger stages were located outside roots until harvest. In a related study, nematodes were isolated from the roots, root surfaces, and soil associated with roots of whole corn and potato plants sampled from the field. Nematode population estimates calculated from the whole plant samples were generally lower than those based on soil cores, but showed similar patterns of population growth. Nematode density per gram dry weight was highest in roots, intermediate on root surfaces, and lowest in soil. Estimates of the absolute abundance of nematodes in each of the three habitats were highest in roots or soil, depending on the sampling date, and lowest on root surfaces. This study demonstrates that P. scribneri inhabits soil environments even when host roots are present and illustrates the importance of considering all possible habitats when estimating the size of Pratylenchus spp. populations.     

Host Suitability of Soybean Cultivars and Breeding Lines to Reniform Nematode in Tests Conducted in 2001

Reproduction of reniform nematode Rotylenchulus reniformis on 139 soybean lines was evaluated in a greenhouse in the summer of 2001. Cultivars and lines (119 total) were new in the Arkansas and Mississippi Soybean Testing Programs, and an additional 20 were submitted by C. Overstreet, Louisiana State Extension Nematologist. A second test of 32 breeding lines and 2 cultivars from the Clemson University soybean breeding program was performed at the same time under the same conditions. Controls were the resistant cultivars Forrest and Hartwig, susceptible Braxton, and fallow infested soil. Five treatment replications were planted in sandy loam soil infested with 1,744 eggs and vermiform reniform nematodes, grown for 10 weeks in 10 cm-diam.- pots. Total reniform nematodes extracted from soil and roots was determined, and a reproductive factor (final population (Pf)/ initial inoculum level (Pi)) was calculated for each genotype. Reproduction on each genotype was compared to the reproduction on the resistant cultivar Forrest (RF), and the log ratio [log₁₀(RF + 1) is reported. Cultivars with reproduction not significantly different from Forrest (log ratio) were not suitable hosts, whereas those with greater reproductive indices were considered suitable hosts. These data will be useful in the selection of soybean cultivars to use in rotation with cotton or other susceptible crops to help control the reniform nematode and to select useful breeding lines as parent material for future development of reniform nematode resistant cultivars and lines.     

Variability in Reproduction of Four Races of Meloidogyne incognita on Two Cultivars of Soybean

Variability in the reproduction of the four races ofMeloidogyne incognita on the soybean cuhivars Pickett 71 and Centennial was studied in growth chamber experiments. Analysis of variance in the number of eggs produced by the races 6 weeks after the plants had been inoculated with 5,000 eggs of each race revealed that the nematode race by soybean cultivar interaction was highly significant (P = 0.001). Races 1, 3, and 4 produced from about 5,000 to 15,000 eggs per root system on Pickett 71 and only from about 300 to 600 eggs per root system on Centennial. In contrast, race 2 produced about 8,000 eggs per root system on Centennial and about 1,200 eggs per root system on Pickett 71. In a second experiment, in which the plants were inoculated with 2,000 second-stage juveniles, race 1 and race 2 produced about 13,000 and 3,000 eggs per root system, respectively, on Pickett 71 and about 600 and 10,000 eggs per root system, respectively, on Centennial. The results suggest that M. incognita resistance in soybean is race-specific.     

Mode of Parasitism of Meloidogyne and Other Nemaiode Eggs by Dactylella oviparasitica

Hyphae of Dactylella oviparasitica proliferated rapidly through MeIoidogyne egg masses, and appressoria formed when they contacted eggs. The fungus probably penetrated egg shells mechanically, although chitinase production detected in culture suggested that enzymatic penetration was also possible. In soil, D. oviparasitica invaded egg masses soon after they were deposited on the root surface and eventually parasitized most of the first eggs laid. Occasionally the fungus grew into Meloidogyne females, halting egg production prematurely. The fungus parasitized eggs in the gelatinous matrix or eggs freed from the matrix and placed on agar or in soil. Specificity in nematode egg parasitism was not displayed, for D. oviparasitica parasitized eggs of four Meloidogyne spp., Acrobeloides sp., Heterodera schachtii, and Tylenchulus semipenetrans. In tests in a growth chamber, parasitism by D. oviparasitica suppressed galling on M. incognita-infected tomato plants.     

Colonization of Soybean Cyst Nematode Females,Cysts, and Gelatinous Matrices by the Fungus Verticillium lecanii

Heterodera glycines was grown in monoxenic culture on soybean roots and then inoculated with the antagonistic fungus Verticillium lecanii. Use of root explant cultures allowed evaluation of the fungus-nematode interaction with the nematode attached to roots or removed from the host, and avoided contamination with other fungi. From 16 hours to 14 days following inoculation, female and cyst samples were examined with the light microscope, or prepared for either conventional or low-temperature scanning electron microscopy. Within 16 hours, hyphae had begun colonizing the gelatinous matrices (GM). The fungus proliferated in the GM of some specimens within a week, but was rarely seen in unhatched eggs. Fungus penetration holes in female and cyst walls were observed 3 days after inoculation; penetration through nematode orifices was not seen at that time. More cysts than females were colonized at the earliest sampling dates. Specimens associated with external hyphae exhibited variable internal colonization, ranging from no fungal penetration to extensive mycelial growth.     

Influence of Herbicide Application to Soybeans on Soybean Cyst Nematode Egg Hatching

The hatching of Heterodera glycines eggs in soybean root exudates collected after postemergence application of three herbicides, and the hatching potential of H. glycines eggs from females feeding on herbicide-treated plants, were measured in vitro. Hatching in all root exudate solutions (RES) was greater than in deionized water but less than in 0.003 M ZnSO₄ solution. Filtering RES with a 0.22-μm-filter increased H. glycines hatching in RES. Application of acifluorfen, bentazon, and lactofen to foliage of soybean plants inhibited hatching of H. glycines eggs from the same plants. Hatching in RES from the different herbicide-treated soybeans was similar. Application of crop oil concentrate and non-ionic surfactant adjuvant to foliage did not affect hatching of H. glycines eggs from soybean plants.