A Role of the Gelatinous Matrix in the Resistance of Root-Knot Nematode (Meloidogyne spp.) Eggs to Microorganisms

The survival of eggs of the root-knot nematode Meloidogyne javanica was studied in a series of experiments comparing the infectivity of egg masses (EM) to that of separated eggs (SE). The EM or SE were placed in the centers of pots containing citrus orchard soil and incubated for 24 hours, 10 days, or 20 days. Following each incubation time, 10-day-old tomato plants were planted in each pot, and 3 to 4 weeks later the plants were harvested and the galling indices determined. In the EM treatments, galling indices of ca. 4.0 to 5.0 were recorded after all three incubation periods; in the SE treatments, the infectivity gradually declined to trace amounts by 20 days. Incubating EM and SE for 2 weeks in four different soil types showed the same pattern in all the soil types: EM caused heavy infection of the test plants while the infection rate from the SE was extremely low. Incubating EM and SE in soil disinfested with formaldehyde resulted in comparable galling indices in most treatments. In petri dish experiments, 100 mg of natural soil was spread at the perimeter of a Phytagel surface and EM or SE of M. incognita were placed in the center. Light microscopy revealed that within 5 to 10 days the SE were attacked by a broad spectrum of microorganisms and were obliterated while the eggs within the EM remained intact. Separated eggs placed within sections of gelatinous matrix (GM) were not attacked by the soil microorganisms. When selected microbes were placed on Phytagel surfaces with EM of M. incognita, electron microscopy demonstrated that at least some microbes colonized the GM. As the major difference between the EM and the SE was the presence of the GM, the GM may serve as a barrier to the invasion of some microorganisms.     

DNA Isolation and GC Base Composition of Four Root-knot Nematode (Meloidogyne spp.) Genomes

Phenol extraction and cesium trifluoroacetate ultracentrifugation were compared for efficiency in the extraction of DNA from eggs and second-stage juveniles of four species of Meloidogyne. The second method proved to be more satisfactory in that it yielded larger amounts of DNA, shortened the extraction period, and reduced sample handling by eliminating phenol and ether extraction and RNAse treatment. It also made possible the extraction of DNA: from more than one sample at a time. The mean base compositions (% GC) of the total DNA of M. incognita, M. javanica, M. arenaria, and M. hapla, as determined by thermal denaturation tests, were quite similar, as they ranged only between 31 and 33%. Similarly, the thermal stability of the DNA of all four species covered a narrow range from 82.97 to 83.63 C.     

Nematicidal Effects of Silver Nanoparticles on Root-knot Nematode in Bermudagrass

Certain nematodes are common soilborne organisms found in turfgrass in the United States that cause significant economic damage to golf course turf. One of the most prevalent plant-parasitic nematodes infesting turfgrass are root-knot nematodes (Meloidogyne spp.). Chemical treatment options for root-knot nematodes in turfgrass are limited, and there is a need for new nematicidal active ingredients to address this problem. In this study, we evaluated the use of silver nanoparticles (AgNP) as a potential nematicide in laboratory and field experiments. AgNP was synthesized by a redox reaction of silver nitrate with sodium borohydride using 0.2% starch as a stabilizer. When J2 of M. incognita were exposed to AgNP in water at 30 to 150 μg/ml, >99% nematodes became inactive in 6 hr. When turfgrass and soil composite samples infested with M. graminis were treated with 150 μg/ml AgNP, J2 were reduced in the soil samples by 92% and 82% after 4- and 2-d exposures, respectively, in the treated compared to the nontreated soil samples. Field trials evaluating AgNP were conducted on a bermudagrass (Cynodon dactylon × C. transvaalensis) putting green infested with M. graminis. Biweekly application of 90.4 mg/m² of AgNP improved turfgrass quality in one year and reduced gall formation in the roots in two years without phytotoxicity. The AgNP application did not significantly reduce the number of M. graminis J2 in plots during the growing season. The laboratory assays attested to the nematicidal effect of AgNP, and the field evaluation demonstrated its benefits for mitigating damage caused by root-knot nematode in bermudagrass.     

Histopathology of Root-knot Nematode (Meloidogyne incognita) Infection on White Yam (Dioscorea rotundata) Tubers

White yam tissues naturally and artificially infected with root-knot nematodes were fixed, sectioned, and examined with a microscope. Infective second-stage juveniles of Meloidogyne incognita penetrated and moved intercellularly within the tuber. Feeding sites were always in the ground tissue layer where the vascular tissues are distributed in the tubers. Giant cells were always associated with xylem tissue. They were thin walled with dense cytoplasm and multinucleated. The nuclei of the giant cells were only half the size of those found in roots of infected tomato plants. Normal nematode growth and development followed giant cell formation. Females deposited eggs into a gelatinous egg mass within the tuber, and a necrotic ring formed around the female after eggs had been produced. Second-stage juveniles hatched, migrated, and re-infected other areas of the tuber. No males were observed from the tuber.     

Description of the Kona Coffee Root-knot Nematode,Meloidogyne konaensis n. sp.

Meloidogyne konaensis n. sp. is described from coffee from Kona on the island of Hawaii. The perineal pattern of the female is variable in morphology, the medial lips of the female are divided into distinct lip pairs, and the excretory pore is 2-3 stylet lengths from the base of the stylet. Mean stylet length is 16.0 μm, and the knobs gradually merge with the shaft. The knobs are indented anteriorly and rounded posteriorly and the dorsal esophageal gland orifice (DEGO) is long, 3.5-7 μm. The morphology of the stylet of the male is the most useful diagnostic character, with 6-12 large projections protruding from the shaft. One medial lip may be divided into distinct lip pairs. A large intestinal caecum often extends nearly to the level of the DEGO. Mean juvenile length is 502 μm, mean stylet length is 13.4 μm, and mean tail length is 58 μm. The tail may be distinctly curved ventrally and the phasmids are located in the ventral incisure about one anal body width posterior to the anus.     

Scanning Electron Microscope Study of the Root-knot Nematode (Meloidogyne incognita) on Tomato Root

This study examines the types of structural information that can be gained by utilizing the scanning electron microscope (SEM) and a cryofracture technique to examine the host-parasite interaction. Roots of tomato, Lycopersicon esculentum cv. Marglobe, were cultured aseptically and inoculated with the root-knot nematode, Meloidogyne incognita. Twenty-four hours to four weeks after inoculation, developing galls were removed from the cultures and processed for SEM observation. The cryofracture technique was used to reveal internal structural features within the developing galls. The results illustrate structural details concerning penetration of the roots, differentiation of syncytia, and development of the nematodes beginning with the second-stage larvae and ending with adult egg-laying females.     

Effect of Gamma-irradiation and Heat on Root-knot Nematode,Meloidogyne javanica

Effects of gamma-irradiation on the root-knot nematode Meloidogyne javanica were investigated. A dose of 7.5 kGy killed all second-stage juveniles (J2) within 1 day after treatment. Egg hatch was completely inhibited at 6.25 kGy. A bioassay on tomato measuring galling and egg production was used to determine the infectivity of irradiated J2 and J2 hatched from irradiated eggs. The J2 and eggs irradiated with a dose of 4.25 kGy did not induce galls or reproduce on tomato plants. When nematodes were exposed to combined irradiation and heat treatment, no synergistic effect on J2 or eggs was measured. Heat treatment at 49° C for 10 minutes or 20 minutes without irradiation immobilized J2 and prevented egg development. Irradiation rates needed to kill or incapacitate M. javanica were high and may be impractical as a quarantine measure.     

Hirsutella rhossiliensisand Verticillium chlamydosporium as Biocontrol Agents of the Root-knot Nematode Meloidogyne hapla on Lettuce

Hirsutella rhossiliensis and Verticillium chlamydosporium infected second-stage juveniles (J2) and eggs of Meloidogyne hapla, respectively, in petri dishes and in organic soil in pots planted to lettuce in the greenhouse. In vitro, H. rhossiliensis produced 78 to 124 spores/infected J2 of M. hapla. The number of J2 in roots of lettuce seedlings decreased exponentially with increasing numbers of vegetative colonies of H. rhossiliensis in the soil. At an infestation of 8 M. hapla eggs/cm³ soil, 1.9 colonies of H. rhossiliensis/cm³ soil were needed for a 50% decrease in J2 penetration of lettuce roots. Egg-mass colonization with V. chlamydosporium varied from 16% to 43% when soil was infested with 8 M. hapla eggs and treated with 5,000 or 10,000 chlamydospores of V. chlamydosporium/cm³ soil. This treatment resulted in fewer J2 entering roots of bioassay lettuce seedlings planted in the infested soils after harvesting the first lettuce plants 7 weeks after infestation with M. hapla. Hirsutella rhossiliensis (0 to 4.3 colonies/cm3 soil), V. chlamydosporium (500 to 10,000 chlamydospores/cm3 soil), or their combination, added to organic soils with 8 M. hapla eggs/cm³ soil, generally did not affect lettuce weight, root galling, or egg production of M. hapla. However, when lettuce was replanted in a mix of infested and uninfested soil (1:3 and 1:7, v:v), egg production was lower in soils with V. chlamydosporium than in soils without the fungus. Both fungi have potential to reduce the M. hapla population, but at densities below 8 eggs/cm³ soil.     

Identification of Sources of Resistance to Four Species of Root-knot Nematodes in Tobacco

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.     

Resistance of Cucumis spp. to the Root-knot Nematode,Meloidogyne incognita acrita

The nature of resistance in Cucumis ficifolius and C. metuliferus to the root-knot nematode, Meloidogyne incognita acrita, was studied under greenhouse conditions. Although as many larvae penetrated the roots of these species as those of the susceptible C. melo, few developed to the adult female stage. Resistance in C. ficifolius and C. metuliferus was associated with hindrance of larval development beyond the second stage, delayed development of larvae to adults and stimulation toward maleness. Tissue necrosis or hypersensitivity was not associated with larval penetration. Comparisons of the histopathology of 26-day-old infections of C. melo and C. metuliferus roots showed no observable differences in the type of giant cell development in regions of roots associated with adult females. However, in C. rnetuliferus immature nematodes were associated with small giant cells which were limited to a few cells near the head of the nematode.     

RKN Lethal DB: A database for the identification of Root Knot Nematode (Meloidogyne spp.) candidate lethal genes

Root Knot nematode (RKN; Meloidogyne spp.) is one of the most devastating parasites that infect the roots of hundreds of plant species. RKN cannot live independently from their hosts and are the biggest contributors to the loss of the world''s primary foods. RNAi gene silencing studies have demonstrated that there are fewer galls and galls are smaller when RNAi constructs targeted to silence certain RKN genes are expressed in plant roots. We conducted a comparative genomics analysis, comparing RKN genes of six species: Meloidogyne Arenaria, Meloidogyne Chitwoodi, Meloidogyne Hapla, Meloidogyne Incognita, Meloidogyne Javanica, and Meloidogyne Paranaensis to that of the free living nematode Caenorhabditis elegans, to identify candidate genes that will be lethal to RKN when silenced or mutated. Our analysis yielded a number of such candidate lethal genes in RKN, some of which have been tested and proven to be effective in soybean roots. A web based database was built to house and allow scientists to search the data. This database will be useful to scientists seeking to identify candidate genes as targets for gene silencing to confer resistance in plants to RKN.

Availability

The database can be accessed from http://bioinformatics.towson.edu/RKN/     

Molecular Diagnosis of Meloidogyne Species

Genetic variation within nuclear and mitochondrial DNA of Meloidogyne species and host races has been evaluated for the development of root-knot nematode molecular diagnostics. This review summarizes the distinctive features of several useful DNA-based assays for plant-parasitic nematodes, focusing upon the direct application of these procedures for Meloidogyne detection, identification, and systematics.     

Meloidogyne petuniae n. sp. (Nemata: Meloidogynidae), a Root-knot Nematode Parasitic on Petunia in Brazil

Meloidogyne petuniae n. sp. is described and illustrated from specimens parasitic on petunia (Petunia hybrida L.) in Brasilia, Brazil. The perineal pattern of the female is elongate to ovoid with a high, squarish arch and widely spaced, coarse striae. The stylet of the female is 12.9-16.5 µm long and has three small, rounded knobs that are distinctly set off from the shaft. Each knob is marked by a deep longitudinal indentation posteriorly and anteriorly. In SEM the base of the shaft appears to be divided into six distinct ridges. The excretory pore opens about 15.4-53.6 µm from the head end. Males are approximately 0.8-2.2 mm long. Most specimens have a high and narrow head cap, but in some the head cap is narrow and low. The stylet of the male is 21.1-26.0 µm long and has small, rounded knobs, set off from the shaft, but not indented as in the female. Second-stage juveniles are 353-464 µm long; the labial disc is fused with the medial lips to form a dumbbell-shaped head cap; the medial lips are indented posteriorly; and the head region is marked with one to two irregular annulations. The stylet is 9.2-10.8 µm long and has rounded, posteriorly sloping knobs. The tail is slender, approximately 46.4-57.2 µm long, and has a short hyaline terminus, 10.3-13.5 µm long. The somatic chromosome number is 2n = 41 and the esterase phenotype is VS1-S1, with S1 being a weak band. The malate dehydrogenase phenotype is N1, which is unique for this species. Petunia, tomato, tobacco, pea, and bean are good hosts; pepper, watermelon, and sweet corn are poor hosts; and peanut, cotton, and soybean are non-hosts. Galls produced by this species are smaller on petunia than on tomato.     

Meloidogyne morocciensis n. sp. (Meloidogyninae), a Root-knot Nematode from Morocco

Meloidogyne morocciensis n. sp. is described from specimens parasitic on peach rootstock from Morocco. This species exhibits a combination of morphological characters similar to M. arenaria, M. incognita, and M. javanica. The perineal pattern of females is oval to squarish with a moderately high to high dorsal arch, and widely spaced, smooth striae; lateral lines are absent. The stylet, 16.5 μm long, has transversely ovoid, set-off knobs. Males have a set-off, annulated head region. The large, rounded labial disc is distinctly demarcated from the crescent-shaped medial lips; lateral lips are absent. The robust stylet, 24.6 μm long, has large, rounded knobs that taper slightly posteriorly. Mean second-stage juvenile (J2) length is 401 μm. The set-offhead region has incomplete annulations; the lip structures are dumbbell shaped. The stylet, 12.3 μm long, has rounded knobs that slope posteriorly. The J2 tail, 52.6 μm long, has irregularly sized annules in the posterior region and ends in a bluntly rounded tip. Tomato, tobacco, pepper, and watermelon are good hosts; cotton and peanut are not hosts. Meloidogyne morocciensis n. sp. reproduces by mitotic parthenogenesis and has a somatic chromosome number of 47-49. Its esterase phenotype is identical with the three-banded phenotype (A3) of M. arenaria.     

Description and SEM Observations of Meloidogyne chitwoodi n. sp. (Meloidogynidae), a Root-knot Nematode on Potato in the Pacific Northwest

Meloidogyne chitwoodi n. sp. is described and illustrated from potato (Solanum tuberosum) originally collected from Quincy, Washington, USA. This new species resembles M. hapla, but its perineal pattern is basically round to oval with distinctive and broken, curled, or twisted striae around and above the anal area. The vulva is in a sunken area devoid of striae. Vesicles or vesicle-like structures are present in the median bulb of females. The larva tail, being short and blunt with a hyaline tail terminal having little or no taper to its rounded terminus, is distinctively different from M. hapla. SEM observations revealed the nature of the perineal pattern and details of the head of larvae and males, and showed the spicules to have dentate tips ventrally. Hosts for M. chitwoodi n. sp. include potato, tomato, corn, and wheat but not strawberry, pepper, or peanut. The latter three crops are excellent hosts for M. hapla. The known distribntion of this new root-knot species presently involves certain areas of Idaho, Washington, and Oregon. The common name "Columbia root-knot nematode" is proposed for M. chitwoodi n. sp.     

Estimating Incidence of Attachment of Pasteuria penetrans Endospores to Meloidogyne spp. with Tally Thresholds

Pasteuria penetrans has .been identified as an important biological control agent of root-knot nematodes. In this study the use of tally thresholds was evaluated for estimating P. penetrans endospore attachment to second-stage juveniles (J2) of Meloidogyne spp. A tally threshold (T) is defined as the maximum number of individuals in a sample unit that may be treated as absent based on binomial sampling. Three different data sets that originated from centrifugal bioassay, incubation bioassay, and field experiments were investigated. The data sets each contained 70, 33, and 111 estimates of the mean number of endospores attached per J2 (m), respectively. Empirical relationships between m and proportions of J2 with ≤T endospores attached (P_T) were developed using parameters from the linear regression of ln(m) on P_T (0 < P_T < 1): ln(m) = a + b P_T, T was set to 0, 1, 2, 3, 4, 5, 8, and 10 endospores/J2. The results indicated that the variances of linear equations tended to decrease with increasing T values for all three data sets. T values of 0, 1, 8, and 10 endospores/J2 for centrifugal bioassay and incubation bioassay, and of 0, 1, 2, and 3 endospores/J2 for field experiments were associated with an r² of >= 0.8. These T values were robust for estimating m from P_T, reducing the variability as well as the time and effort spent in estimating the mean number of endospores attached per J2.     

Meloidogyne grahami n. sp. (Meloidogynidae), A Root-knot Nematode on Resistant Tobacco in South Carolina

Meloidogyne grahami n. sp. is described and illustrated from specimens on tobacco (Nicotiana tabacum) originally from Florence, South Carolina. Considered for several years to be only a race of M. ineognita, this new species readily attacks NC-95 tobacco, a variety with resistance to the M. incognita group that is common in the major U.S. tobacco-producing areas. M. grahami n. sp. is related most closely to the three subspecies of the M. incognita group but differs from all of them, especially in its distinctive perineal pattern and larger larvae (av. 421 μm, vs. 385 μm or less). Also, the dorsal esophageal gland orifice of females of M. grahami n. sp. is further from the base of the styler (5 μm) than in M. i. incognita and M. i. acrita. Comments are given on the distribution of this new species.     

Meloidogyne mayaguensis n. sp. (Meloidogynidae), a Root-knot Nematode from Puerto Rico

Meloidogyne mayaguensis n. sp. is described and illustrated from specimens obtained from galled roots of eggplant, Solanum melongena L., from Puerto Rico. The perineal pattern of females is round to ovoid with fine, widely spaced striae. It has occasional breaks of striation laterally and a circular tail tip area lacking striae. The stylet, 15.8 μm long, has reniform knobs that merge gradually with the stylet shaft. Males have a high, rectangular, smooth head region, not set off from the body contour. The labial disc is continuous with the medial lips which do not slope posteriorly. The styler, 22.9 μm long, has large rounded backward sloping knobs; the shaft is of uneven diameter. Mean body length of second-stage juveniles is 453.6 μm. The truncate head region is not annulated, and the rounded, slightly raised labial disc and the crescentic medial lips form dumbbell-shaped lip structures. The stylet, 11.6 μm long, has rounded, posteriorly sloping knobs. The slender tail, 54.4 μm long, gradually tapers to a bluntly pointed tip. Tomato, tobacco, pepper, and watermelon are good hosts; cotton and peanut are not hosts. M. mayaguensis n. sp. reproduces by mitotic parthenogenesis and has a somatic chromosome number of 2n = 44-45. The enzyme patterns are unique among Meloidogyne species.     

Extremely Sensitive Thermotaxis of the Nematode Meloidogyne incognita

Eggs of the root-knot nematode Meloidogyne incognita were acclimated to 23 C. Newly hatched second-stage juveniles migrated toward higher temperatures when placed in shallow thermal gradients averaging 23 C. The threshold gradient for this response was below 0.001 C/cm, with a best estimate of 4 x 10⁻⁴ C/cm. Calculations of physical limitations on thermotaxis indicate that this sensitivity is well within the limits of what is physically possible.     

Intra- and Interpopulation Genome Variation In Meloidogyne arenaria

The genetic heterogeneity of two M. arenaria race 2 populations (designated Pelion and Govan) was examined using RFLP analysis of 12 clonal lines established from single egg masses (six distinct clonal lines from each population). These populations are essentially identical by traditional biochemical and race identification schemes; however, the Govan population is more aggressive than the Pelion population, producing larger galls and exhibiting greater reproductive capabilities on many soybean cultivars and experimental accessions. Variation at the genomic DNA level was examined using probes representative of expressed DNA sequences present in the eukaryotic genome. Ribosomal DNA, interspersed repeated sequences, and cDNA probes were tested for detection of polymorphism within and between single egg mass lines of each population. Cloned cDNAs and ribosomal intergenic spacer sequences detect polymorphism both within and between populations, demonstrating the usefulness of these sequence classes for molecular genetic analysis of population structure and genome evolution.