First reports of Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae (Tylenchomorpha) from Greece and further records of four other nematode species.

Nematode samplings in various areas and crops of Greece were carried out and the recovered nematode species were characterized using morphological and molecular data. Seven species of plant-parasitic nematodes were recovered, three of which are reported for the first time in Greece, including Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae. Four other recovered species had already been reported in Greece, including Bitylenchus hispaniensis, Helicotylenchus microlobus, Nanidorus minor and Scutellonema brachyurus. D2-D3 segments of 28S rRNA gene for all of these nematode species are provided.

Nematicidal Potential of Thymol against Meloidogyne javanica (Treub) Chitwood.

Root-knot nematodes (RKN; Meloidogyne spp.) are obligatory endoparasites with worldwide distribution which cause severe damage to agricultural crops. The present study aimed to define the nematicidal activity of thymol on different life stages of the root-knot nematode Meloidogyne javanica (Treub) Chitwood, at concentrations of 37.5-1000 µL/L. This study is the first to report the effect of thymol on egg differentiation and also its vapor and sublethal concentration activities. A mortality of greater than 90% of M. javanica second-stage juveniles (J2s) occurred after 96 h of exposure at a concentration of 500 µL/L. At this concentration, thymol inhibited 59.7% of nematode hatching. In addition, the use of thymol at sublethal concentrations reduced the number of females per gram in tomato roots in a pot test, as well as inhibiting egg differentiation. On the contrary, no nematostatic effects were observed in paralysis bioassays. The results presented here indicate that the use of thymol may show its potential as a source of a new sustainable nematicidal product.

Comparison of a Vintage and a Recently Released Nematicide for the Control of Root-Knot Nematodes and Side Effects on Two Entomopathogenic Nematodes.

Root-knot nematodes can cause tremendous losses in vegetable crops. Farmers usually rely on synthetic nematicides to protect their crops. Recently, newly released nematicides are giving farmers an alternative in chemical control for nematodes. In the present study, the efficacy of vintage nematicide was compared to that of a relatively new nematicide, fluopyram. The latter was always more effective in substantially lower concentrations than oxamyl. Fluopyram paralyzed more than 80% J2s after 24 h immersion at the concentration of 0.25 µL L-1, while the percentage was increased close to 100% after immersion for 48 and 96 h. Similar levels of dead J2s were observed after immersion of J2s in oxamyl at concentrations higher than 8 µL L-1 (24 and 48 h) or 4 µL L-1 (96 h). An evident decrease of egg differentiation was observed when fluopyram concentration was increased to 8 µL L-1, while no significant decrease in egg differentiation was recorded at any concentration of oxamyl. Egg hatching was decreased at concentrations of fluopyram higher than 4 µL L-1, while no reduction was observed even when the concentration of oxamyl was increased to 64 µL L-1. The efficacy of fluopyram in soil was superior compared to that of oxamyl. For the first time, the systemic action of fluopyram is recorded in trials with tomato plants. On the other hand, compared to oxamyl, fluopyram seems to be more toxic to non-target organisms such as the entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora.

The Use of Essential Oil and Hydrosol Extracted from Cuminum cyminum Seeds for the Control of Meloidogyne incognita and Meloidogyne javanica.

The essential oil (EO) and hydrosol (HL) isolated from Cuminum cyminum (cumin) seeds were evaluated against the root-knot nematodes Meloidogyne incognita and M. javanica. The efficacy of extracts on the motility, hatching, and survival in soil of second-stage juveniles (J2s), and the activity on egg differentiation were tested. All J2s were paralyzed after immersion in the EO at 62.5 µL/L concentration for 96 h. Encouraging results were recorded using HL equal to or higher than 10% concentration for both Meloidogyne species tested. More than 70% paralyzed J2s were recorded after immersion for 48 h, while the percentage was increased to higher than 90% after 96 h of immersion. A clear effect on egg differentiation was observed after immersion in EO or HL. A significant decrease in egg differentiation was revealed at even low concentrations of EO while an evident decrease in egg differentiation was recorded after immersion of eggs in 50% HL dilution. Decreased hatching of M. incognita and M. javanica J2s was observed with the increase in concentration. The lowest numbers of hatched J2s were recorded when EO was used at 1000 and 2000 µL/L concentrations. A constant reduction in root-knot nematode J2 hatching was observed upon increasing the concentration of HL from 5% to 50%. The EO of C. cyminum is characterized by the presence of γ-terpinene-7-al (34.95%), cumin aldehydes (26.48), and α-terpinene-7-al (12.77%). The above constituents were observed in HL following the same order as that observed in EO. The components γ-terpinene (11.09%) and ο-cymene (6.56%) were also recorded in EO while they were absent in HL.

The Use of Essential Oil and Hydrosol Extracted from Satureja hellenica for the Control of Meloidogyne incognita and M. javanica.

Essential oil (EO) and hydrosol (HL) isolated from an indigenous plant species Satureja hellenica were evaluated against Meloidogyne incognita and M. javanica. Particularly, the activity of extracts on a second stage juvenile's (J2s) motility, the hatching of J2s from eggs, egg differentiation and the effect on J2s in soil were tested. A paralysis of 100% of the J2s of both species was recorded after 96 h of immersion in the essential oil, at a dose of 2000 µL/L. At the same dose, the percentage of paralyzed J2s after 48 h of immersion was more than 80%, for both Meloidogyne species. The use of hydrosol has shown encouraging results only in the dilution of 50%, where for both Meloidogyne species tested, the percentage of paralyzed J2s was more than 70% after 48 h of immersion, while the percentage was increased to 90% after 96 h of immersion. Egg differentiation was ceased after immersion, either in EO or HL. However, this decrease in egg differentiation was evident only at higher concentrations of EO and at the highest HL dilution (0.5 v/v). The hatching of M. incognita J2s was decreasing as the dose was increasing. The lowest numbers of hatched J2s were recorded at the doses of 2000 and 4000 µL/L. A clear reduction in M. javanica J2s hatching was observed as the dose was increased to 250 µL/L, a fact constantly observed as the dose was increasing up to 4000 µL/L. Lower numbers of nematodes were recorded in roots grown in infested soil after the application of EO or HL at the highest doses. The EO of S. hellenica is characterized by the presence of p-cymene (27.46%) and carvacrol (23.25%), and in a lesser extent of other constituents, such as borneol (6.79%), carvacrol methylether (6.77%), γ-terpinene (4.63%) and 4-terpineol (3.65%). Carvacrol was the major constituent found in the HL (50.12%), followed by borneol and 4-terpineol (20.42 and 6.72%, respectively).

The potential of eugenol as a nematicidal agent against Meloidogyne javanica (Treub) Chitwood.

Root-knot nematodes (RKN; Meloidogyne spp.) are the most destructive plant parasites in vegetable production and their control is very challenging. This study aimed to define the nematicidal activity of eugenol on different life stages at 33.75 to 1,000 ppm doses against the root-knot nematode Meloidogyne javanica (Treub) Chitwood, 1949. This work is the first to report the effect of eugenol on egg differentiation and its vapor and sublethal doses activities. Second-stage juveniles (J2) were dead (99.5-100%) after 48 hr of exposure at a dose of 500 ppm. At this concentration, eugenol inhibited more than 70% nematode hatching. Additionally, the use of eugenol at sublethal doses reduced the number of females per gram in tomato roots in a pot test, and also inhibited egg differentiation. To the contrary, no nematostatic effects were observed in nematode motility bioassays. The phenolic monoterpenoid eugenol described herein merits further study as potential nematicide against the rootknot nematode Meloidogyne javanica.

Synergistic and antagonistic interactions of terpenes against Meloidogyne incognita and the nematicidal activity of essential oils from seven plants indigenous to Greece.

BACKGROUND: Biorational means for phytonematode control were studied within the context of an increasingly ecofriendly pest management global approach. The nematicidal activity and the chemical composition of essential oils (EOs) isolated from seven plants grown in Greece and ten selected compounds extracted from them against second-stage juveniles (J2) of Meloidogyne incognita (Kof. & White) Chitwood were evaluated using juvenile paralysis experiments. Additionally, synergistic and antagonistic interactions between nematicidal terpenes were studied using an effect addition model, with the comparison made at one concentration level. RESULTS: The 96 h EC(50) values of Foeniculum vulgare Mill., Pimpinella anisum L., Eucalyptus meliodora A Cunn ex Schauer and Pistacia terebinthus L. were 231, 269, 807 and 1116 µg mL(-1) , respectively, in an immersion bioassay. Benzaldehyde (9 µg mL(-1) ) was the most toxic compound, followed by γ-eudesmol (50 µg mL(-1) ) and estragole (180 µg mL(-1) ), based on 96 h EC(50) values. The most potent terpene pairs between which synergistic actions were found, in decreasing order, were: trans-anethole/geraniol, trans-anethole/eugenol, carvacrol/eugenol and geraniol/carvacrol. CONCLUSION: This is the first report on the activity of F. vulgare, P. anisum, E. meliodora and P. terebinthus, and additionally on synergistic/antagonistic nematicidal terpene interactions, against M. incognita, providing alternative methods for nematode control.

Phytochemistry and nematicidal activity of the essential oils from 8 Greek Lamiaceae aromatic plants and 13 terpene components.

Eight essential oils (EOs) as well as 13 single terpenes were studied for their nematicidal activity against Meloidogyne incognita , for three immersion periods (24, 48, and 96 h). The EOs were isolated from eight Greek Lamiaceae species: Melissa officinalis , Sideritis clandestina , Origanum dictamnus , Ocimum basilicum , Mentha pulegium , Origanum vulgare , Vitex agnus castus , and Salvia officinalis . The EOs nematicidal activity was correlated to their chemical composition as well as to the pure terpenes' activity tested individually. Clear dose and time response relationships were established. The EOs of O. vulgare, O. dictamnus, M. pulegium, and M. officinalis exhibited high nematicidal activity against M. incognita, and the EC(50) values (96 h) were calculated at 1.55, 1.72, 3.15, and 6.15 muL/mL, respectively. The activity of the nematicidal terpenes was found to decrease in the order l-carvone, pulegone, trans-anethole, geraniol, eugenol, carvacrol, thymol, terpinen-4-ol, and the respective EC(50) values (24 h) were calculated in the range of 115-392 mug/mL. Terpenes tested individually were more active than as components in EO, implementing antagonistic action.

Factors affecting the efficacy of non-fumigant nematicides for controlling root-knot nematodes.

Second-stage juveniles (J2) and egg masses of root-knot nematodes as well as root debris heavily infected by the latter were exposed for different periods of time to six different doses of the nematicides cadusafos and fenamiphos. The efficacy of the nematicides increased significantly with increasing exposure time. Both nematicides were more effective against J2, although they could not provide acceptable control of J2 inside egg masses or heavily galled root debris. The effect of different application strategies on the efficacy and persistence of certain nematicides was also assessed in a field study. Cadusafos, fenamiphos, fosthiazate and oxamyl were applied in field micro-plots either as a single full dose at the time of crop establishment or as multiple reduced-rate applications at 14-day intervals throughout the cropping period, and their efficacy and persistence were determined using bioassays and analytical studies. Fosthiazate was the most efficient nematicide studied, and this was mainly attributed to its long soil persistence. Oxamyl also provided adequate nematode control for the first 48-56 days after its application, regardless of the application method used and its relatively rapid field dissipation. Fenamiphos and cadusafos failed to provide adequate nematode control, although cadusafos was the most persistent of the nematicides tested. The failure of fenamiphos to provide adequate nematode control was mainly attributed to its rapid degradation by soil micro-organisms, which were stimulated after its repeated low-rate application at 14-day intervals. In contrast cadusafos failure was attributed to the inability of the nematicide to reduce nematode populations even at relatively high concentrations in soil.

The enhanced biodegradation of fenamiphos in soils from previously treated sites and the effect of soil fumigants.

The application of fenamiphos either alone or in combination with soil fumigants is a common practice in greenhouses and potato-cultivation areas in Greece. However, repeated applications of fenamiphos in the same field for a number of years can lead to the development of enhanced biodegradation of the nematicide. Studies in previously treated greenhouse sites and potato field sites in Greece were employed in order to investigate the development of enhanced biodegradation of fenamiphos and the respective effect of soil fumigants on the development of the phenomenon. Enhanced biodegradation of fenamiphos in a soil from a previously treated greenhouse site from the area of Aggelohori in Northern Greece was observed using both incubation and bioassay studies with nematodes. Fumigation of the enhanced soil with methyl bromide (MeBr) only temporarily inhibited degradation of fenamiphos unlike metham sodium (MS) whose application significantly reduced microbial degradation of fenamiphos. Similarly, enhanced biodegradation of fenamiphos was evident in soil from potato fields that had a history of previous exposure to fenamiphos. The slow rates of fenamiphos degradation observed in soils from the previously treated sites after sterilization with broad-spectrum antibiotics and also in soils from previously untreated sites suggested that soil microorganisms were responsible for its rapid degradation. The inhibition of enhanced biodegradation of fenamiphos in soil from the previously treated greenhouse site caused by the antibiotic penicillin probably indicates that Gram+ or other bacteria sensitive to penicillin are responsible for the rapid degradation of fenamiphos in this soil. No cross-adaptation was observed between fenamiphos and other nematicides registered in Greece for the control of root-knot and potato cyst nematodes, including cadusafos, ethoprophos, and oxamyl. According to our results, applications of MS followed by fenamiphos or in rotation with other registered nematicides are the most promising means for minimizing the risk of development of enhanced biodegradation of fenamiphos in soils.

Evaluation of chemical and integrated strategies as alternatives to methyl bromide for the control of root-knot nematodes in Greece.

Current environmental awareness has led to a greater demand for alternative nematode control strategies. Three field experiments were established to compare management tactics on cucumber in commercial greenhouses naturally infested with root-knot nematodes (Meloidogyne spp). Cucumber rootstocks which have shown resistance to soil-borne diseases were tested to reveal any resistance/tolerance to root-knot nematodes, and integration of these rootstocks with nematicides was investigated. Metham-sodium and 1,3-dichloropropene (1,3-D) provided good control of nematode populations when their application was followed by the application of a non-fumigant nematicide such as cadusafos or oxamyl. Neither fumigant could provide season-long control of nematode populations, and a further application of cadusafos was required for satisfactory control. The efficacy of metham-sodium was significantly increased when injected into soil in comparison with its application through the drip irrigation system. The use of rootstocks resistant to soil-borne fungal pathogens used together with chemical means of nematode control provided promising results for their further use in integrated strategies as alternatives to methyl bromide. However, the latter was the superior treatment for the control of root-knot nematodes in soil infested with residues of galled roots. Dazomet, metham-sodium nor the non-fumigant nematicides oxamyl and fenamiphos could reduce nematode population as efficiently as methyl bromide. None of the chemicals tested except methyl bromide could enter galled roots and kill surviving nematodes.

Chemical alternatives to methyl bromide for the control of root-knot nematodes in greenhouses.

The complete phase-out of methyl bromide from use in developed countries by 1 January 2005 will cause many problems in agricultural industries that are now heavily reliant on its use. Three field experiments were established to compare management tactics on tomato and cucumber in commercial greenhouses naturally infested with root-knot nematodes (Meloidogyne spp). Reduction of nematode juveniles in soil and roots to nil detection levels was observed in all plots following soil fumigation with methyl bromide. A significant reduction of nematode juveniles and root-galling index was observed in plots treated with metham-sodium, dazomet and 1,3-dichloropropene compared with the control and plots treated with non-fumigant nematicides. Reduction of the nematode population led to an increase in fruit yield. However, data collected from the second cultivation season indicated that single control methods such as fumigant or contact nematicides alone cannot drastically decrease initial nematode population and those nematodes which escape control lead to population increase by the end of the cropping season.