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.     

Influence of Six Vegetable Cultivars on Reproduction of Meloidogyne javanica

Replicated field and greenhouse experiments were used to evaluate the effect of tomato, cabbage, cucumber, carrot, Amaranthus hybridus, and pepper on growth and fecundity of Meloidogyne spp., particularly M. javanica. In the field tests, tomato, cucumber, and carrot favored population increases of Meloidogyne spp., while Amaranthus, pepper, and cabbage limited them. Some cropping sequences that included crops from the latter group had a suppressive effect on population growth. Thus, of the 36 cropping sequences that were investigated, the following kept the pests in check: tomato-pepper; tomato-Amaranthus; cabbage-pepper; Amaranthus-pepper; carrot-cabbage; pepper-pepper; pepper-Amaranthus; and Amaranthus-pepper. In the greenhouse tests, tomato, cucumber, and carrot had a high number of galls per 50 cm of root, large, conspicuous galls and egg masses, and a high number of larvae per egg mass. Thus, they were highly susceptible. Cabbage and Amaranthus were unsuitable hosts as reflected in the absence of galls or a low number per 50 cm of root. small size of galls and egg masses, and few progeny on the subsequent crop of pepper. The length of time required for eggs to hatch on different hosts varied considerably and is thought to be a significant factor in infection of hosts.     

Parasitism of Meloidogyne eggs by a new fungal parasite

Dactylella oviparasitica, a fungus isolated from Meloidogyne egg masses, was shown to parasitize eggs on agar and in soil, when inoculated as either myceliunt or conidia. The fungus also grew saprophytically on eggs killed ,with methyl bromide or heat. The amount of parasitism in the laboratory suggested that the fungus may be a useful biological control agent against Meloidogyne.     

A Method for Field Infestation with Meloidogyne incognita

A field inoculation method was developed to produce Meloidogyne spp. infestation sites with minimal quantities of nematode inoculum and with a reduced labor requirement compared to previous techniques. In a preseason-methyl bromidefumigated site, nematode egg suspensions were delivered at concentrations of 0 or 10^x eggs/m of row where x = 2.12, 2.82, 3.52, or 4.22 through a drip line attached to the seed firmer of a commercial 2-row planter into the open seed furrow while planting cowpea. These treatments were compared to a hand-inoculated treatment, in which 10^3.1 eggs were delivered every 30 cm in 5 ml of water agar suspension 2 weeks after planting. Ten weeks after planting, infection of cowpea roots was measured by gall rating and gall counts on cowpea roots. A linear relationship between the inoculation levels and nematode-induced galls was found. At this time, the amount of galling per root system in the hand-inoculated treatment was less than in the machine-applied treatments. Advantages of this new technique include application uniformity and low population level requisite for establishing the nematode. This method has potential in field-testing of Meloidogyne spp. management strategies by providing uniform infestation of test sites at planting time.     

Effects of temperature on the life-history traits of Sancassania (Caloglyphus) berlesei (Acari: Astigmatina: Acaridae) feeding on root-knot nematodes,Meloidogyne spp. (Nematoda: Meloidogynidae)

Sancassania (Caloglyphus) berlesei (Michael) is a cosmopolitan and free-living mite that inhabits soil as well as laboratory colonies of insects and fungi and may have a role as a biocontrol agent of nematodes. In this study, we investigated the effects of temperature on the development, reproduction, and food consumption of S. berlesei fed egg masses of root-knot nematodes, Meloidogyne spp., an important group of agricultural pests. Mites were reared at 20, 25 or 30 °C in the dark. The mites could feed on the nematode egg masses, and their developmental time decreased at higher temperatures. Time from the egg to adult was similar in females and males reared at the same temperature. Adult females lived longer than males at 25 °C, but not at 20 or 30 °C. Generally, females showed a higher rate of food consumption than males. Females laid the largest number of eggs at 20 and 25 °C (199.7 and 189.8 eggs/female, respectively), but the intrinsic rate of natural increase was highest at 30 °C (r _m = 0.29). In comparing our data with previous reports, we noted that S. berlesei that fed on egg masses of root-knot nematodes showed a longer developmental time and a lower reproductive rate than Sancassania mites that fed on other diets. Nonetheless, the relatively high value of r _m (e.g., at 25 and 30 °C) suggests that this mite may have certain advantages as a biocontrol agent of root-knot nematodes.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Species of Root-knot Nematodes and Fungal Egg Parasites Recovered from Vegetables in Almería and Barcelona,Spain

Intensive vegetable production areas were surveyed in the provinces of Almería (35 sites) and Barcelona (22 sites), Spain, to determine the incidence and identity of Meloidogyne spp. and of fungal parasites of nematode eggs. Two species of Meloidogyne were found in Almería—M. javanica (63% of the samples) and M. incognita (31%). Three species were found in Barcelona, including M. incognita (50%), M. javanica (36%), and M. arenaria (14%). Solanaceous crops supported larger (P < 0.05) nematode numbers than cucurbit crops in Almería but not in Barcelona. Fungal parasites were found in 37% and 45% of the sites in Almería and Barcelona, respectively, but percent parasitism was never greater than 5%. Nine fungal species were isolated from single eggs of the nematode. The fungi included Verticillium chlamydosporium, V. catenulatum, Fusarium oxysporum, F. solani, Fusarium spp., Acremonium strictum, Gliocladium roseum, Cylindrocarpon spp., Engiodontium album, and Dactylella oviparasitica. Two sterile fungi and five unidentified fungi also were isolated from Meloidogyne spp. eggs.     

Effect of Meloidogyne incognita and Importance of the Inoculum on the Yield of Eggplant

The relationship between population densities of race 1 of Meloidogyne incognita and yield of eggplant was studied. Microplots were infested with finely chopped nematode-infected pepper roots to give population densities of 0, 0.062, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, and 128 eggs and juveniles/cm³ soil. Both plant growth and yield were suppressed by the nematode. A tolerance limit of 0.054 eggs and juveniles/cm³ soil and a minimum relative yield of 0.05 at four or more eggs and juveniles/cm³ soil were derived by fitting the data with the equation y = m + (1 - m)z^P⁻T. Maximum nematode reproduction rate was 12,300. Hatch of eggs from egg masses in water or from sodium hypochlorite dissolved egg masses was similar (41% and 39%), but egg viability was significantly greater from egg masses in water (58%) than from sodium hypochlorite dissolved egg masses (12%) after 4 weeks. Greater numbers of nematodes were collected from roots of tomatoes from soil infested with entire egg masses than from tomato roots from soil infested with egg masses dissolved by sodium hypochlorite.     

Fungi associated with eggs and first-instar larvae of the European corn borer

Fungi isolated from field-collected egg masses of the European corn borer, Ostrinia nubilalis, were identified as Alternaria spp., A. porri, Fusarium spp., Fusarium oxysporum, Beauveria bassiana, Mucor spp., and an unidentified yeast. Most fungi were associated with predator injury to the egg mass. Bioassay of fungi on egg masses, however, showed that Alternaria spp. and A. porri reduced the hatch of both injured and uninjured egg masses, and Mucor sp. reduced the hatch only when the egg mass was injured.     

Detection and biocontrol potential of <Emphasis Type="Italic">Verticillium leptobactrum</Emphasis> parasitizing <Emphasis Type="Italic">Meloidogyne</Emphasis> spp.

Three isolates of Verticillium leptobactrum proceeding from egg masses of root-knot nematodes (RKN) Meloidogyne spp. and soil samples collected in Tunisia were evaluated against second-stage juveniles (J2) and eggs of M. incognita, to determine the fungus biocontrol potential. In vitro tests showed that V. leptobactrum is an efficient nematode parasite. The fungus also colonized egg masses and parasitized hatching J2. In a greenhouse assay with tomato plants parasitized by M. incognita and M. javanica, V. leptobactrum was compared with isolates of Pochonia chlamydosporia and Monacrosporium sp., introducing the propagules into nematode-free or naturally infested soils. The V. leptobactrum isolates were active in RKN biocontrol, improving plants growth with a significant increase of tomato roots length, lower J2 numbers in soil or egg masses, as well as higher egg mortalities. In a second assay with M. javanica, treatments with three V. leptobactrum isolates reduced egg masses on roots as well as the density of J2 and the number of galls. To evaluate the fungus capability to colonize egg masses a nested Real-time polymerase chain reaction (PCR) assay, based on a molecular beacon probe was used to assess its presence. The probe was designed on a V. leptobactrum ITS region, previously sequenced. This method allowed detection of V. leptobactrum from egg masses, allowing quantitative DNA and fungal biomass estimations.     

Optimum Initial Inoculum Levels for Evaluation of Resistance in Tomato to Meloidogyne spp. at Two Different Soil Temperatures

The effects of Meloidogyne incognita or M. javanica at five initial inoculum levels of 20, 100, 200, 1,000, and 2,000 eggs and infective juveniles per seedling on ''Floradade,'' ''Nemarex,'' ''Patriot,'' and ''PI 129149-2(sib)-5'' tomatoes maintained at 25 or 32.5 C were studied. The number of egg masses on roots of the susceptible cultivar Floradade was similar for both species of root-knot nematodes at either 2.5 or 32.5 C soil temperatures. At 25 C, very low numbers of egg masses were produced by both species of root-knot nematodes on Nematex, Patriot, and Lycopersicon peruvianum PI 129149-2(sib)-5. At 32.5 C, the best inoculum level for assessing resistance in these tomato genotypes was 200 eggs and infective juveniles per seedling. With 28 days of incubation, this temperature and inoculum level produced quantitative differences in resistance for both species of Meloidogyne.     

Suppression of Meloidogyne incognita and M. javanica by Pasteuria penetrans in Field Soil

The role of Pasteuria penetrans in suppressing numbers of root-knot nematodes was investigated in a 7-year monocuhure of tobacco in a field naturally infested with a mixed population of Meloidogyne incognita race 1 and M. javanica. The suppressiveness of the soil was tested using four treatments: autoclaving (AC), microwaving (MW), air drying (DR), and untreated. The treated soil bioassays consisted of tobacco cv. Northrup King 326 (resistant to M. incognita but susceptible to M. javanica) and cv. Coker 371 Gold (susceptible to M. incognita and M. javanica) in pots inoculated with 0 or 2,000 second-stage juveniles of M. incognita race 1. Endospores of P. penetrans were killed by AC but were only slightly affected by MW, whereas most fungal propagules were destroyed or inhibited in both treatments. Root galls, egg masses, and numbers of eggs were fewer on Coker 371 Gold in MW, DR, and untreated soil than in AC-treated soil. There were fewer egg masses than root galls on both tobacco cultivars in MW, DR, and untreated soil than in the AC treatment. Because both Meloidogyne spp. were suppressed in MW soil (with few fungi present) as well as in DR and untreated soil, the reduction in root galling, as well as numbers of egg masses and eggs appeared to have resulted from infection of both nematode species by P. penetrans.     

Relationship Between Egg Viability and Population Densities of Meloidogyne incognita on Cotton

Cotton seedlings were inoculated with a range of initial populations (Pi) of Meloidogyne incognita in greenhouse experiments to test the relationship between nematode population densities and egg viability. In two of three experiments, a significant (P < 0.05) negative linear relationship was detected between percentage of hatch of first generation eggs and log Pi. A similar relationship between hatch and root-gall index was observed. In two experiments numbers of eggs judged to be nonviable based on appearance were significantly greater (P < 0.05) in the highest Pi (60,000 eggs/seedling) treatments than in treatments with lower Pi (600-6,000 eggs/seedling). It was concluded that Pi affects egg viability measured as percentage of hatch and that this relationship may play a role in the density-dependent winter survival rates of Meloidogyne species.     

Correlation of Tobacco Yield,Value, and Root-Knot Index with Early-to-Midseason,and Postharvest Meloidogyne Population Densities

Certain nematicidal treatments for the control of root knot of tobacco in six field experiments in North Carolina were used to determine early, midseason, and postharvest densities of eggs and larvae of Meloidogyne spp., postharvest root-knot indices, and crop yield and value. Various statistical correlations showed that population densities determined 6-8 wk after transplanting were more indicative of treatment effectiveness than postharvest densities. Logarithmic transformation of early and midseason population data stabilized the variance.     

Effects of Tagetes patula on Active and Inactive Stages of Root-Knot Nematodes

Although marigold (Tagetes patula) is known to produce allelopathic compounds toxic to plant-parasitic nematodes, suppression of Meloidogyne incognita can be inconsistent. Two greenhouse experiments were conducted to test whether marigold is more effective in suppressing Meloidogyne spp. when it is active rather than dormant. Soils infested with Meloidogyne spp. were collected and conditioned in the greenhouse either by 1) keeping the soil dry (DRY), 2) irrigating with water (IRR), or 3) drenching with cucumber (Cucumis sativus) leachate (CL) for 5 wk. These soils were then either planted with cucumber, marigold or remained bare for 10 wk. Suppression of nematode by marigold was then assayed using cucumber. DRY conditioning resulted in the highest number of inactive nematodes, whereas CL and IRR had higher numbers of active nematodes than DRY. At the end of the cucumber bioassay, marigold suppressed the numbers of Meloidogyne females in cucumber roots if the soil was conditioned in IRR or CL, but not in DRY. However, in separate laboratory assays, marigold root leachate slightly reduced M. incognita J2 activity but did not reduce egg hatch (P > 0.05). These finding suggest that marigold can only suppress Meloidogyne spp. when marigold is actively growing. This further suggests that marigold will more efficiently suppress Meloidogyne spp. if planted when these nematodes are in active stage.     

Broad Meloidogyne Resistance in Potato Based on RNA Interference of Effector Gene 16D10

Root-knot nematodes (Meloidogyne spp.) are a significant problem in potato (Solanum tuberosum) production. There is no potato cultivar with Meloidogyne resistance, even though resistance genes have been identified in wild potato species and were introgressed into breeding lines. The objectives of this study were to generate stable transgenic potato lines in a cv. Russet Burbank background that carry an RNA interference (RNAi) transgene capable of silencing the 16D10 Meloidogyne effector gene, and test for resistance against some of the most important root-knot nematode species affecting potato, i.e., M. arenaria, M. chitwoodi, M. hapla, M. incognita, and M. javanica. At 35 days after inoculation (DAI), the number of egg masses per plant was significantly reduced by 65% to 97% (P < 0.05) in the RNAi line compared to wild type and empty vector controls. The largest reduction was observed in M. hapla, whereas the smallest reduction occurred in M. javanica. Likewise, the number of eggs per plant was significantly reduced by 66% to 87% in M. arenaria and M. hapla, respectively, compared to wild type and empty vector controls (P < 0.05). Plant-mediated RNAi silencing of the 16D10 effector gene resulted in significant resistance against all of the root-knot nematode species tested, whereas R_Mc1(blb), the only known Meloidogyne resistance gene in potato, did not have a broad resistance effect. Silencing of 16D10 did not interfere with the attraction of M. incognita second-stage juveniles to roots, nor did it reduce root invasion.     

Susceptibility of different developmental stages of the root-knot nematode, Meloidogyne incognita, to the nematicide oxamyl

Oxamyl (6.25 μg ml^-1 soil water) was applied to cucumber roots containing Meloidogyne incognita at different stages of nematode development. Oxamyl was more effective in reducing the proportion of juveniles which developed into females when applied soon after infection to second stage juveniles than when applied later (to third and fourth stage juveniles). Early application of oxamyl also significantly reduced the proportion of females with egg masses, whereas late application had no such effect. However, the number of eggs per egg mass and the size of the young adult females was significantly reduced by all oxamyl treatments - the earlier the application the greater the effect. These results support the hypothesis that actively feeding second stage juveniles of Meloidogyne spp. are more susceptible to systemic nematicides than are the non-feeding third and fourth stage juveniles.     

Optimization of Mitochondrial DNA-based Hybridization Assays to Diagnostics in Soil

Nucleic acid hybridization among root-knot nematode mitochondrial DNAs can be used to identify several Meloidogyne species. Research was initiated to optimize mitochondrial DNA-based molecular diagnostics for the demanding environments likely to be encountered in field isolates. DNA hybridization using reconstituted DNA-soil mixtures revealed a loss of assay sensitivity ranging from 34% to 92% with four agronomic soils tested. This problem was alleviated by the addition of exogenously added DNA. Variation in nematode egg lysis procedures also affected hybridization efficiency, with NaOC1 treatment most effective at disrupting Meloidogyne eggs. These optimized conditions permit detection of mtDNA released from one to five Meloidogyne eggs using standard nucleic acid hybridization procedures.     

Susceptibility of Nezara viridula (L.) (Hemiptera: Pentatomidae) Egg Masses of Different Sizes to Parasitism by Trissolcus basalis (Woll.) (Hymenoptera: Platygastridae) in the Field

Egg masses of Nezara viridula (L.) are commonly parasitized by Trissolcus basalis (Woll.), and we investigated the role of size of egg masses on parasitization by T. basalis. Sentinel egg masses were exposed to parasitism in the field for 6–7 days, when they were collected for evaluation of parasitoid emergence. We recorded the number of eggs per egg mass, the number of emerged hosts, and the number of empty and parasitized eggs. We calculated the proportion of attacked host egg masses (DE), the proportion of parasitized eggs per attacked egg mass (PE), and total parasitism (PI). The total number of egg masses exposed to parasitism was 330. The minimum, mean, and maximum egg mass sizes were 25, 75.2, and 111, respectively. DE and PE varied widely between different fields, and they were independent of egg mass size. In 14.2% of all parasitized egg masses, we found simultaneous emergence of T. basalis and N. viridula independently of host egg mass size. PE exhibited low variability compared with PI and DE, which were linearly related. PI and DE values from other field studies are consistent with the linear relationship, suggesting that PI is mostly related to the proportion of the DE. This also suggests that total parasitism is independent of egg mass size, of possible differences in plant species, and T. basalis density and strains.     

Recovery and Longevity of Egg Masses of Meloidogyne incognita during Simulated Winter Survival

Effects of soil matrix potential on longevity of egg masses of Meloidogyne incognita were determined during simulated winter conditions. Egg masses were recovered from isolated root fragments incubated in field soil at matrix potentials of -0.1, -0.3, -1.0, and -4.0 bars throughout winter survival periods of 10 weeks for tomato roots and 12 weeks for cotton roots. Egg masses were more superficial on cotton roots than on tomato roots and were more easily dislodged from cotton roots during recovery of root fragments by elutriation. The rate of decline in numbers of eggs and J2 per egg mass was greater in wet as compared to dry soils (P = 0.01), with the relationship between numbers of eggs and J2 per egg mass and time being best described by quadratic models. Percentage hatch of recovered eggs declines linearly with time at soil matrix potentials of -0.1 and -0.3 bars, but at -1.0 and -4.0 bars the percentage hatch of recovered eggs increased before declining. Effects of soil matrix potential on numbers of eggs per egg mass and percentage hatch of recovered eggs were consistent with previous reports that low soil moisture inhibits egg hatch before affecting egg development. Estimations of egg population densities during winter survival periods will be affected by ability to recover infected root fragments from the soil without dislodging associated egg masses. There is a need for procedures for extraction of egg masses not attached to roots from the soil.