Evaluating Pochonia chlamydosporia in a double-cropping system of lettuce and tomato in plastic houses infested with Meloidogyne javanica

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1 Plant Pathology (2003) 52, Blackwell Publishing Ltd. Evaluating Pochonia chlamydosporia in a double-cropping system of lettuce and tomato in plastic houses infested with Meloidogyne javanica S. Verdejo-Lucas a *, F. J. Sorribas b, C. Ornat b and M. Galeano a a Departamento Protección Vegetal, IRTA, Carretera de Cabrils, Cabrils, Barcelona; and b Departamento Ingeniería Agroalimentaria y Biotecnología, Universitat Politècnica de Catalunya, Comte d Urgel 181, Barcelona, Spain The effect of Pochonia chlamydosporia, a facultative fungal parasite of nematode eggs, alone or in combination with oxamyl was evaluated in a double-cropping system of lettuce and tomato in unheated plastic houses infested with Meloidogyne javanica at two sites for two consecutive growing seasons. An additional treatment of methyl bromide fumigation was included to compare crop yield in nematode-free vs. nematode-infested soil. Final population densities, reproductive rate, root gall rating, and egg production were determined after each crop. Pochonia chlamydosporia was isolated from nematode eggs up to nine months after application to soil. The fungus survived in the rhizosphere for the entire growing season at one site, but only at low densities. Final population densities of M. javanica decreased after cultivation of lettuce and increased after tomato, and this pattern of population fluctuation was unaffected by treatment, experiment or site. The reproductive rate on lettuce was equal to or below 1, and it was similar among treatments in both experiments at both sites. Eggs were not found on lettuce roots. On tomato, the reproductive rate in the fungus + oxamyl treatment was significantly lower (P < 0 05) than other treatments in experiment 1 at both sites. Fungus + oxamyl consistently reduced root gall ratings on tomato in all cases, but numbers of eggs per g root varied depending on treatment. Methyl bromide-treated plots remained free of M. javanica at the end of the 2-year study. Keywords: biological control, crop rotation, methyl bromide, oxamyl, root-knot nematodes Introduction Vegetable crops are intensively grown through most of the year in many areas of the Mediterranean region, where two to three crops are usually cultivated at the same site from spring through winter. Tomato and lettuce are the most frequently cultivated vegetables for fresh market, and are grown in plastic houses in the coastal areas of north-eastern Spain (Sorribas & Verdejo-Lucas, 1994). Root-knot nematodes have become increasingly important in plastic houses due to the damage they cause, and because of their rapid spread in several countries of the Mediterranean region including Spain (Ornat et al., 1999). Currently, control of root-knot nematodes is based mainly on the used of soil fumigants, and organophosphate and carbamate nematicides. In the near future, however, growers will have to adopt alternative management *To whom correspondence should be addressed. soledad.verdejo@irta.es Accepted 13 February 2003 strategies due to loss of broad-spectrum soil fumigants. These strategies have so far received little attention for nematode control due to the availability of reliable broadspectrum soil fumigants such as methyl bromide (Roberts, 1993). Methyl bromide, widely used in intensive production systems, is being replaced by other soil fumigants such as 1 3-dichloropropene and metham sodium. Management strategies for nematode control in Mediterranean climates include crop rotation, soilless cultivation, organic soil amendments, resistant cultivars, nematode antagonists, steam, soil solarization and chemical control (Verdejo-Lucas, 1999). The strategies can be used singly, in combination or sequentially, and their effect should be considered over multiple crop cycles to determine their short- and long-term effectiveness. The combination of two or more of these strategies will probably be needed to mitigate nematode problems, as there is seldom a single effective method. Pochonia chlamydosporia (syn. Verticillium chlamydosporium) is a facultative parasite of nematode eggs, and has been extensively investigated as a potential biological control agent for cyst and root-knot nematodes (Kerry & 2003 BSPP 521

2 522 S. Verdejo-Lucas et al. Jaffee, 1997). The fungus is widely distributed in agricultural soils, and can survive as a saprophyte in the absence of a nematode host. It has recently been isolated from eggs of Meloidogyne spp. associated with vegetable crops in Spain (Verdejo-Lucas et al., 2002). The objective of this study was to evaluate the effect of P. chlamydosporia alone or in combination with oxamyl on final population densities of M. javanica, on damage caused by the nematode, and on crop yield in a double-cropping system of lettuce and tomato. Materials and methods Two experiments (Exp. 1 and Exp. 2) were conducted in two unheated plastic houses located at the Centre de Cabrils, Institut de Recerca i Tecnologia Agroalimentàries, Cabrils, Barcelona (site Q21), and at the Experimental Farm Can Comas, Consell Comarcal del Baix Llobregat, El Prat, Barcelona, Spain (site CC) for two consecutive growing seasons. The first season (November 1998 July 1999) constituted Exp. 1, and the second (October 1999 July 2000), Exp. 2. The soil at site Q21 was a sandy loam with 85 8% sand, 8 1% silt and 6 1% clay, ph 8 1, 0 9% organic matter (w/w), and 0 40 ds m 1 electric conductivity. The soil at site CC was a loam with 46 5% sand, 41% silt and 12 5% clay, ph 8 1, 2 4 organic matter and 0 58 ds m 1 electric conductivity. The soil of both plastic houses was infested with M. javanica, and preplanting densities ranged from 42 to 1750 secondstage juveniles (J 2 ) per 250 cm 3 soil (mean ± SD 510 ± 410) at site CC, and from 250 to 1240 J 2 per 250 cm 3 soil (685 ± 390) at site Q21. Lettuce, Lactuca sativa type Maravilla cv. Arena, and tomato, Lycopersicon esculentum cv. Durinta, were grown on rotation in autumn winter and spring summer, respectively, for two consecutive years starting in November The experimental design consisted of randomized incomplete blocks with five replicated plots per treatment. The individual treatment units (plots) were 9 or 12 5 m 2 at sites CC and Q21, respectively, and there were 10 lettuce plants, or 2 9 tomato plants m 2 in each plot. Treatments included P. chlamydosporia (Pc) applied to lettuce; oxamyl applied to tomato; a combination of the two previous treatments (Pc + oxamyl); and untreated control consisting of plots infested with M. javanica. Oxamyl as Vydate L 24% a.i. (Du Pont Ibérica, Barcelona) was applied as a soil drench using a watering can 3 and 21 days after planting tomato at a rate of 7 5 L ha 1. The soil was irrigated immediately after application of the nematicide. An additional treatment was included consisting of methyl bromide fumigation to compare crop yield in nematode-free vs. nematode-infested soil. Methyl bromide (98% methyl bromide + 2% chloropicrin) (Ribas, Mataró, Barcelona) was applied at a rate of 75 g m 2 under a plastic seal in October The integrity of each plot was maintained over both seasons. Soil temperature at 15 cm depth was 17 and 21 C at sites CC and Q21, respectively, at the time of fumigant application. Fungal inoculum, densities of P. chlamydosporia and egg parasitism Isolate ICARVc-10 of P. chlamydosporia (provided by B.R. Kerry, Rothamsted Research, Harpenden, UK) was used. The inoculum was prepared by the Departamento de Recursos Naturales, University of Alicante, Alicante, Spain, and was mass-produced in 300 ml conical flasks containing a moist, autoclaved mixture of coarse sand (3 4 mm particles) and cornflour (1 : 1 v/v). After 4 weeks incubation at 25 C, the cultures were washed through 250 and 53 µm sieves to remove sand and corn, and the fungal propagules, mainly chlamydospores, were collected on a 10 µm sieve. Fungal biomass was mixed with fine sand as a carrier to bulk up the inoculum. The concentration of chlamydospores was estimated in diluted samples using a haemocytometer (de Leij et al., 1993). To check the viability of the chlamydospores, 1 g of the inoculum was diluted in 9 ml of a 0 05% agar solution and 200 µl of dilution 10 2 and 10 3 plated on 9 cm Petri dishes containing sorbose (2 g L 1 ), agar (12 g L 1 ) and antibiotics (50 mg L 1 each of streptomycin sulphate, chloramfenicol and chlortetracycline). The percentage of germinated chlamydospores was estimated after incubation at 25 C for 2 days. The fungus was applied at a rate of chlamydospores per plant in Exp. 1. Aliquots (60 cm 3 ) of the sand chlamydospore mixture were added into the planting hole at the time of transplanting lettuce. In Exp. 2, the fungus was applied to lettuce at a rate of chlamydospores per plant by removing the first 15 cm of soil from the planting row, mixing it thoroughly with the inoculum in a concrete mixer, and returning the mixture to the planting row. Soil fungus densities were quantified from inoculated plots 4 weeks after planting lettuce; at the end of the lettucegrowing period; and before and after growing tomato. Individual samples consisted of nine soil cores taken from the first 30 cm of soil with a soil auger (2 5 cm diameter). A subsample of 1 g from each plot was used for dilution plating on a semiselective medium (de Leij & Kerry, 1991). Colonies of P. chlamydosporia on plates were counted after incubation at 25 C for 3 weeks. After harvesting, a 1 g representative subsample from fresh, chopped roots was crushed in 9 ml 0 05% agar solution using a sterile pestle and mortar, and 10 2 and 10 3 dilution series were prepared. Aliquots of each dilution were plated onto a semiselective medium to determine P. chlamydosporia in the rhizosphere. Parasitism of nematode eggs was assessed following the procedure described by de Leij & Kerry (1991). Aliquots of the egg suspension were spread onto three replicated Petri dishes containing a restrictive growth medium (Lopez-Llorca & Duncan, 1986). Eggs were considered parasitized if fungal hyphae were growing out from them after 48 h incubation at 25 C. Parasitism was confirmed by transferring parasitized eggs individually to cornmeal agar medium. Cultures were observed for the presence of chlamydospores 1 week after incubation at 25 C.

3 Evaluating biological and chemical control of Meloidogyne 523 Densities of M. javanica and evaluation of nematode damage Composite soil samples were collected at the beginning (P i ) and end (P f ) of the lettuce- and tomato-growing periods in each experiment at each site. Individual samples consisted of nine soil cores taken from the first 30 cm of soil with a soil auger (2 5 cm diameter). Soil cores were mixed thoroughly and nematodes extracted from 500 cm 3 soil subsamples using Baermann trays. Juveniles migrating to the water were collected 1 week later, concentrated on a 25 µm sieve, and counted. The number of J 2 s was expressed per 250 cm 3 of soil. The reproduction rate for each crop was calculated as the relationship P f /P i based on soil counts of second-stage individuals (J 2 ). Following sampling for final densities (P f ), 10 (Exp. 1) or eight (Exp. 2) randomly selected lettuce or tomato plants per plot were uprooted and rated for galling on a scale 0 10, where 0 = complete and healthy root system and 10 = plants and roots dead (Zeck, 1971). Roots from each plot were then combined and chopped for egg extraction. Eggs were extracted from three 5 g (lettuce) or 10 g (tomato) root subsamples by blender maceration in a 0 5% NaOCl solution for 10 min (Hussey & Barker, 1973). The number of eggs was expressed per g fresh root tissue. Soil temperatures at each site were recorded daily at 30 min intervals with temperature probes places at 15 cm depth. Mean daily soil temperatures at sites CC and Q21 are provided in Fig. 1. The number of degree-days accumulated by M. javanica was calculated using a base temperature of 13 C, and 343 C as the minimum thermal time requirement for one generation (Trudgill, 1995; Tzortzakakis & Trudgill, 1996). Crop yield Lettuces were allowed to grow until they reached marketable size, and then harvested. Yield was assessed by determining the fresh weight of 10 (Exp. 1) or eight (Exp. 2) randomly selected lettuce heads per plot. The same plants were used for root gall rating. To determine tomato yield, fruits produced per 10 (Exp. 1) or eight plants (Exp. 2) per plot were harvested as they matured. Tomatoes were harvested once each week for 6 weeks. The number of fruits was counted, and they were weighed. The cumulative yield was expressed as kg m 2. Figure 1 Mean daily soil temperature at 15 cm depth from November 1998 to August 2000 in a plastic house cultivated with lettuce in autumn winter and tomato in spring summer for two consecutive growing seasons at site CC in El Prat, and at site Q21 in Cabrils, Barcelona, Spain.

4 524 S. Verdejo-Lucas et al. Crop management Lettuce was planted in November 1998 and October 1999, and harvested in February of 1999 and 2000 in Exp. 1 and Exp. 2, respectively. Tomato was cropped from March to July in 1999 and 2000, and harvested after 4 months growth in both experiments in July 1999 and Plants received drip irrigation as needed, and were fertilized weekly with a fertilizer solution consisting of NPK ( ), and iron chelate and micronutrients at rates of 31 and 0 9 kg ha 1, respectively. The ingredients were mixed in a tank and delivered through the irrigation system, except in Exp. 1 at site CC, where it was broadcast. Soil preparation between successive crops was done by hand-hoeing plots individually to prevent cross-contamination between treatments. Tomato plants were vertically trained using canes, and plants used for yield assessment were marked 4 weeks after transplanting. Tomato was pollinated by a colony of Bombus bees placed in each plastic house at first blossom. After the final tomato harvest, plants were cut at ground level and, once dried, removed from the plastic house to prevent nematode populations from increasing further. Statistical analysis Data on the number of J 2 in soil, reproductive index (P f /P i ) and eggs per g root were transformed to [log(x + 1)] and subjected to anova using the GLM procedure of SAS version 8 (SAS Institute Inc., Cary, NC, USA). When the overall F-test was significant, means were separated by the least significant difference (LSD) method (P < 0 05). Data on yield of lettuce and tomato were subjected to anova and analysed by experiment and site. Again, when the overall F-test was significant, means were separated by the LSD procedure (P < 0 05). Results Densities of P. chlamydosporia and egg parasitism Native strains of P. chlamydosporia were not detected in soil samples collected at either plastic house before starting the study. Chlamydospore germination from the inoculum prepared for both experiments was greater than 80%, thus chlamydospores were considered viable. Fungal abundance was variable. Thus 5 2% parasitism was recorded from two of the plots treated with the fungus alone in Exp. 2 at site CC, but parasitized eggs were not detected in Exp. 1. At site Q21, parasitism was 1% in Exp. 1, and ranged from 3 to 5 3% in Exp. 2, where parasitized eggs were recorded from all the plots that received the fungus either alone or in combination with oxamyl. In contrast, parasitism was recorded only from two (fungus alone) and three plots (Pc + oxamyl) in Exp. 1. The numbers of colony-forming units (cfu) per g soil decreased progressively from the time of application until the end of the study at both sites. Nevertheless, P. chlamydosporia was recovered from tomato root samples 9 and 8 months after application of fungus to the preceding crop in Exp. 1 and Exp. 2, respectively, at site Q21. In contrast, P. chlamydosporia was not recovered from tomato roots at site CC. Densities of M. javanica and evaluation of nematode damage Final soil densities of M. javanica on lettuce did not differ among treatments in any of the experiments conducted at either plastic house (Tables 1 and 2). Galled roots occurred on lettuce in Exp. 2 but not in Exp. 1 at both sites. At site CC, lettuce roots showed a similar gall rating in all treatments, and the percentage of plants with galled roots ranged from 22 to 45%. At site Q21, gall rating in fungustreated plots was lower (P < 0 05) than in the remaining nematode-infested ones in Exp. 2 (Table 2). The percentage of lettuce with galled roots was 29, 38, and 66% in plots treated with Pc + oxamyl, fungus alone, or left untreated, respectively. The nematode did not produce eggs on lettuce (Tables 1 and 2). Final densities, reproductive rate (P f /P i ), gall rating and egg production were reduced (P < 0 05) on tomato by Pc + oxamyl in Exp. 1 at site CC (Table 1). The percentage of tomatoes with galled roots ranged from 42 to 90%, in plots treated with Pc + oxamyl or left untreated, respectively. In Exp. 2, Pc + oxamyl and oxamyl alone also reduced gall rating and egg production (Table 1). Final densities were lowest (P < 0 05) in the oxamyl treatment, but they only differed from plots treated with the fungus alone (Table 1). At site Q21, Pc + oxamyl reduced the P f /P i relationship on tomato in Exp. 1, and root galling in both experiments (Table 2). Despite differences in gall rating, 100% of the tomato plants showed galled roots in both experiments at site Q21. The nematicide had a greater effect on M. javanica in the loamy soil of site CC than in the sandy loam soil of site Q21, and it was more effective in Exp. 1 than Exp. 2. The nematode was not detected in methyl bromidetreated plots at either site at the end of the 2-year study. Crop yield Lettuce showed similar head weight (g per plant) irrespective of treatment at both plastic houses. Cumulative tomato yield at site CC was unaffected by oxamyl alone or methyl bromide fumigation in either experiment (Fig. 2). In Exp. 1, yield in Pc + oxamyl was lower (P < 0 05) than in methyl bromide, oxamyl alone, or in plots left untreated, but there were no yield differences in Exp. 2. At site Q21, cumulative tomato yield in nematode-free (fumigated) plots was higher (P < 0 05) than in nematode-infested ones in both experiments (Fig. 2). Significant differences were shown from the first week of harvest in Exp. 1, and after the third week in Exp. 2. The number of fruits per plant and their average fruit weight was higher (P < 0 05) in nematode-free than in nematode-infested plots in Exp. 1 (data not shown). In these plots, the average fruit weight in plots treated with the fungus alone or with Pc + oxamyl

5 Evaluating biological and chemical control of Meloidogyne 525 Table 1 Final population densities, reproductive rate (P f /P i ), gall rating, and eggs per g root of Meloidogyne javanica on lettuce and tomato in a plastic house used for evaluating Pochonia chlamydosporia (Pc) for two consecutive growing seasons at El Prat (site CC), Barcelona, Spain Crop Treatments Juveniles per Gall 250 cm 3 soil P f /P i rating a Eggs per g root b Exp. 1 Lettuce Untreated 189 ± 261 a 0 5 ± 0 6 a 0 0 Pc c 65 ± 47 a 0 8 ± 0 8 a 0 0 Pc + oxamyl d 91 ± 63 a 1 1 ± 0 9 a 0 0 Oxamyl e 84 ± 101 a 0 8 ± 1 1 a 0 0 Tomato Untreated 7900 ± 9130 a 54 ± 43 a 2 0 ± 1 3 a 5100 ± 4050 a Pc 2998 ± 3340 ab 57 ± 59 a 1 5 ± 1 1 b 3710 ± 2960 a Pc + oxamyl 520 ± 670 b 7 ± 8 b 0 4 ± 0 6 d 835 ± 730 b Oxamyl 4086 ± 3635 a 153 ± 189 a 0 9 ± 0 9 c 3120 ± 2850 a Exp. 2 Lettuce Untreated 11 ± 16 a 0 1 ± 0 2 a 0 6 ± 0 6 a 0 Pc 9 ± 15 a 0 6 ± 1 3 a 0 8 ± 0 6 a 0 Pc + oxamyl 10 ± 15 a 0 4 ± 0 5 a 0 6 ± 0 5 a 0 Oxamyl 9 ± 16 a 0 1 ± 0 2 a 0 7 ± 0 6 a 0 Tomato Untreated 360 ± 290 ab f 3 2 ± 1 7 a 1326 ± 836 a Pc 1480 ± 890 a 3 4 ± 1 3 a 867 ± 764 a Pc + oxamyl 290 ± 380 ab 0 8 ± 1 1 c 140 ± 107 b Oxamyl 220 ± 310 b 1 7 ± 1 7 b 530 ± 667 b Values are means ± SD of five replicated plots per treatment. Values within crop and experiment in the same column followed by different lower-case letters are significantly different (P < 0 05). a Based on a scale from 0 (none) to 10 (severe). b Only healthy eggs included. c Applied at time of planting lettuce in November 1998 (Exp. 1) and October 1999 (Exp. 2). d P. chlamydosporia applied with lettuce and oxamyl applied to tomato. e A liquid formulation applied 3 and 21 days after transplanting tomato. f Nematode under detectable levels at planting. Table 2 Final population densities, reproductive rate (P f /P i ), gall rating, and eggs per g root of Meloidogyne javanica on lettuce and tomato in a plastic house used for evaluating Pochonia chlamydosporia (Pc) for two consecutive growing seasons at Cabrils (site Q21), Barcelona, Spain Crop Treatments Juveniles per Gall 250 cm 3 soil P f /P i rating a Eggs per g root b Exp. 1 Lettuce Untreated 330 ± 40 a 0 3 ± 0 1 a 0 0 Pc c 490 ± 290 a 0 5 ± 0 3 a 0 0 Pc + oxamyl d 930 ± 680 a 0 7 ± 0 4 a 0 0 Oxamyl e 360 ± 130 a 0 5 ± 0 2 a 0 0 Tomato Untreated ± a 61 ± 59 a 7 3 ± 1 0 a ± b Pc ± a 98 ± 50 a 7 5 ± 0 7 a ± a Pc + oxamyl ± 6360 a 32 ± 12 b 6 9 ± 0 8 b ± 8600 b Oxamyl ± a 65 ± 40 a 6 3 ± 0 9 c ± ab Exp. 2 Lettuce Untreated 240 ± 130 a 0 1 ± 0 1 a 1 4 ± 1 4 a 0 Pc 280 ± 125 a 0 1 ± 0 1 a 0 4 ± 0 5 b 0 Pc + oxamyl 540 ± 120 a 0 2 ± 0 1 a 0 3 ± 0 6 b 0 Oxamyl 310 ± 170 a 0 1 ± 0 1 a 1 4 ± 1 3 a 0 Tomato Untreated ± 4360 a 23 ± 11 a 7 0 ± 0 7 a ± a Pc ± a 13 ± 10 a 6 1 ± 0 5 b ± a Pc + oxamyl ± 9660 a 10 ± 9 a 5 6 ± 0 7 c ± a Oxamyl ± a 22 ± 11 a 6 7 ± 0 6 a ± a Values are means ± SD of five replicated plots per treatment. Values within crop and experiment in the same column followed by different lower-case letters are significantly different (P < 0 05). a Based on a scale from 0 (none) to 10 (severe). b Only healthy eggs included. c Applied at the time of planting lettuce in November 1998 (Exp. 1) and October 1999 (Exp. 2). d P. chlamydosporia applied with lettuce and oxamyl applied to tomato. e A liquid formulation applied 3 and 21 days after transplanting tomato.

6 526 S. Verdejo-Lucas et al. Figure 2 Cumulative tomato yield in nematode-free and Meloidogyne javanica-infested plots treated with the egg parasitic fungus Pochonia chlamydosporia (Pc), alone or in combination with oxamyl, in two plastic houses in Barcelona, Spain. Mean separation within experiment was assessed by the LSD test (P < 0 05). Plots free of nematodes were obtained by methyl bromide soil fumigation at a rate of 75 g m 2 in October Different lower-case letters beside bars within experiments and within sites denote significant differences. was lower (P < 0 05) than in those without the fungus but treated with oxamyl alone or left untreated. In Exp. 2, the number of fruits per plant was also higher (P < 0 05) in nematode-free than in nematode-infested plots. The Pc + oxamyl treatment produced a lower (P < 0 05) number of fruits than the oxamyl alone or plots left untreated. The average fruit weight was highest (P < 0 05) in nematodefree plots and lowest in those receiving the fungus alone. Yield increase in response to oxamyl did not occur at site Q21 (Fig. 2). Discussion The isolation of P. chlamydosporia from parasitized eggs on tomato roots 8 and 9 months after application of fungus indicated that the fungus survived throughout the growing season, was pathogenic on M. javanica, and was compatible with the agronomic practices and environmental conditions of intensive agriculture in plastic houses. However, abundance of the fungus was low after two applications over four crops. Abundance tended to be greater in the second season (Exp. 2) than in the first (Exp. 1). Several applications might be required for establishment of the fungus in the field because its abundance in the soil or rhizosphere decreases following its incorporation in soil (Kerry et al., 1993; Bourne et al., 1996; Stirling & Smith, 1998; Viaene & Abawi, 2000). Combining the fungus with other control methods may enhance nematode control. Thus the Pc + oxamyl treatment consistently reduced root gall ratings on tomatoes in both experiments and plastic houses, and reduced egg production at site CC. The receptivity of the soil to an antagonist is another factor that may affect the success of biological control methods based on soil application of an antagonistic organism (Rodríguez-Kabana & Morgan-Jones, 1988). Apparently, the sandy soil of site Q21 was more favourable to the fungus and nematode than the loamy sand of site CC, as both organisms were more widely distributed and abundant at site Q21 than site CC despite similar nematode density levels at the beginning of the study, and number of fungus applications. Also, the microflora associated with Meloidogyne egg masses may have an antagonistic effect on P. chlamydosporia (Kok et al., 2001). Soil temperature appeared to play an important role in the nematode fungus interaction. The average soil temperature during the lettuce crop at site CC was 10 6 C (range C) in Exp. 1 and 12 9 C ( C) in Exp. 2; at site Q21 it was 12 8 C ( C) in Exp. 1 and 15 3 C (11 24 C) in Exp. 2. These particular cropping temperatures may have affected fungal development. Pochonia chlamydosporia can grow between 15 and 30 C, but lower temperatures greatly reduce its rate of development. Application of the fungus at optimal temperatures for its development could improve establishment and survival of the fungus. The population dynamics of M. javanica followed a similar pattern of fluctuation in both growing seasons and sites. Densities decreased after lettuce in autumn winter and increased after tomato in spring summer. This pattern was independent of treatment, experiment or site, and was regulated by changes in soil temperature which greatly affected nematode development. The nematode did not accumulate enough heat units to complete one generation on lettuce in autumn winter. A low level of nematode reproduction was expected on lettuce, and in turn increased abundance of the fungus in the rhizosphere (Bourne & Kerry, 1999), but this did not occur. Mean daily soil temperatures in lettuce in the first season were below the temperature threshold of 16 C for root penetration of Meloidogyne (Roberts et al., 1981) for the entire crop cycle at both plastic houses. However, temperatures above this threshold occurred for 22 and 33 days at site CC and Q21, respectively, in the second lettuce crop planted in October 1999 (Exp. 2), which explained root galling in Exp. 2 but not in Exp. 1. After that time, temperatures dropped below 16 C until harvest in February In plastic houses of north-eastern Spain, the average survival rate of Meloidogyne in fallow soils is 0 61 (Ornat et al., 1999), which is within the range observed in Exp. 1, suggesting that population decline after lettuce was similar to that in fallowed soils in this experiment. In Exp. 2, however, the survival rate was lower than that reported;

7 Evaluating biological and chemical control of Meloidogyne 527 lettuce acted as a trap crop as the nematode invaded the root (as shown by root galling), but failed to reproduce. Lettuce was not damaged by M. javanica in either experiment or plastic house. On the contrary, the nematode accumulated enough heat units in spring summer to complete three nematode generations on tomato in both experiments and sites. Soil temperatures were above 16 C for the entire crop cycle on tomato. The nematode reached very high densities on tomato, producing profuse root galling and yield losses at site Q21. It also increased after tomato at site CC, but produced moderate root galling and did not cause yield losses. Temperature increases during the short fallow periods between successive crops (Fig. 1), particularly after tomato, were probably due to withholding irrigation after termination of the crop. None of the treatments produced an increase in yield of lettuce, and only methyl bromide did so on tomato at site Q21, with increases of 50 and 25% in the first and second seasons, respectively. There was no yield increase in response to oxamyl in this study, and it seems that such a response depends on the frequency of multiple applications of the nematicide, and on the soil texture (Garabedian & Van Gundy, 1985; Philis, 1994; Noling & Gilreath, 2000). The overall increase in tomato yield observed in Exp. 2 was possibly due to delivery of the fertilizer through the drip irrigation system instead of broadcasting it. In this study, chemical and biological methods were evaluated alone or in sequence in a double-cropping system of lettuce and tomato. The results showed that this system maintained low population densities, which prevented nematode damage and crop losses at site CC, but not at site Q21. Methyl bromide soil fumigation provided effective and lasting control of M. javanica at both sites. Pochonia chlamydosporia in combination with oxamyl, and oxamyl alone, reduced nematode damage but had no effect on final populations. The fungus alone prevented nematode damage in some situations but did not affect nematode densities. The use of the fungus to control rootknot nematodes has shown promising results in pot and small-scale experiments (Godoy et al., 1983; de Leij et al., 1993; Bourne & Kerry, 1999; Viaene & Abawi, 2000), but limited success when tested in larger-scale field trials (Stirling & Smith, 1998; Tzortzakakis, 2000). It is concluded that effective control of Meloidogyne in plastic houses on a short-term basis will be very difficult to achieve in warm winter climates such as those in the Mediterranean region in soils conducive to the nematode by means other than soil fumigation, as large increases in population density occur in spring summer despite a sharp decrease in autumn winter. Management of rootknot nematodes on a longer-term basis should involve a combination of different control methods such as tillage, resistant cultivars, nonhost plants or trapping crops to reduced population densities to sustainable levels. Knowledge of the population dynamics and ecology of the nematode and its antagonists is important in helping to determine the rational use and best performance of the fungal parasite, and management strategies for nematode control. Acknowlegements This research was funded by the European Union FAIR project CT and Ministerio de Ciencia y Tecnología CICYT project AGF We thank the Consell Comarcal del Baix Llobregat for allowing us to use the Experimental Farm at Can Comas. References Bourne JM, Kerry BR, Effect of the host plant on the efficacy of Verticillium chlamydosporium as a biological control agent of root-knot nematodes at different nematode densities and fungal application rates. Soil Biology and Biochemistry 31, Bourne JM, Kerry BR, De Leij FAAM, The importance of the host plant on the interaction between root-knot nematodes (Meloidogyne spp.) and the nematophagous fungus, Verticillium chlamydosporium Goddard. Biocontrol Science and Technology 6, Garabedian S, Van Gundy SD, Effects of non-fumigant nematicides applied through low-pressure drip irrigation on control of Meloidogyne incognita on tomatoes. Plant Disease 69, Godoy G, Rodríguez-Kábana R, Morgan-Jones G, Fungal parasites of Meloidogyne arenaria eggs in an Alabama soil. A mycological survey and greenhouse studies. Nematropica 13, Hussey RS, Barker KR, A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Disease 57, Kerry BA, Jaffee BA, Fungi as biological control agents for plant parasitic nematodes. In: Wicklow DT, Söderström B, eds. The Mycota IV Environmental and Microbial Relationships. New York, USA: Springer-Verlag, Kerry BR, Kirkwood IA, de Leij FAAM, Barba J, Leijdens MB, Brookes PC, Growth and survival of Verticillium chlamydosporium Goddard, a parasite of nematodes in soil. Biocontrol Science and Technology 3, Kok CJ, Papert A, Hok A, Hin CH, Microflora of Meloidogyne egg masses: species composition, population density and effect on the biocontrol agent Verticillium chlamydosporium (Goddard). Nematology 3, de Leij FAAM, Kerry BR, The nematophagous fungus Verticillium chlamydosporium as a potential biological control agent for Meloidogyne arenaria. Revue de Nématologie 14, de Leij FAAM, Kerry BR, Dennehy JA, Verticillium chlamydosporium as a biological control agent for Meloidogyne incognita and M. hapla in pot and micro-plot tests. Nematologica 39, Lopez-Llorca LV, Duncan JM, New media for the estimation of fungal infection in eggs of the cereal cyst nematode, Heterodera avenae Woll. Nematologica 32, Noling JW, Gilreath JP, Methyl bromide: progress and problems identifying alternatives. Citrus & Vegetable Magazine, Ornat C, Verdejo-Lucas S, Sorribas FJ, Tzortzakakis EA, Effect of fallow and root destruction on survival of root-knot and root-lesion nematodes in intensive vegetable cropping systems. Nematropica 29, 5 16.

8 528 S. Verdejo-Lucas et al. Philis J, Use of dazomet and oxamyl for controlling the root-knot nematode Meloidogyne in glass houses. Nematologia Mediterranea 22, Roberts PA, The future of nematology: integration of new and improved management strategies. Journal of Nematology 25, Roberts PA, Van Gundy SD, McKinney HE, Effects of soil temperature and planting date of wheat on Meloidogyne incognita reproduction, soil populations, and grain yield. Journal of Nematology 13, Rodríguez-Kabana R, Morgan-Jones G, Potential for nematode control by mycofloras endemic in the tropics. Journal of Nematology 20, Sorribas FJ, Verdejo-Lucas S, Survey of Meloidogyne spp. in tomato production fields of Baix Llobregat county, Spain. Journal of Nematology 26, Stirling GR, Smith LJ, Field test of formulated products containing either Verticillium chlamydosporium or Arthrobotrys dactyloides for biological control of root-knot nematodes. Biological Control 11, Trudgill DL, An assessment of the relevance of thermal time relationships to nematology. Fundamental and Applied Nematology 18, Tzortzakakis EA, The effect of Verticillium chlamydosporium and oxamyl on the control of Meloidogyne javanica on tomatoes grown in a plastic house in Crete, Greece. Nematologia Mediterranea 28, Tzortzakakis EA, Trudgill DL, A thermal time-based method for determining the fecundity of Meloidogyne javanica in relation to modeling its population dynamics. Nematologica 42, Verdejo-Lucas S, Nematodes. In: Albajes R, Gullino ML, Van Lanteren JC, Elad Y, eds. Integrated Pest and Disease Management in Greenhouse Crops. Dordrecht, The Netherlands: Kluwer Academic, Verdejo-Lucas S, Ornat C, Sorribas FJ, Stchigel A, Species of root-knot nematodes and fungal egg parasites recovered from vegetables in Almería and Barcelona, Spain. Journal of Nematology 34, Viaene NM, Abawi GS, Hirsutella rhosiliensis and Verticillium chlamydosporium as biological control agents of the root-knot nematode Meloidogyne hapla on lettuce. Journal of Nematology 32, Zeck WM, A rating scheme for field evaluation of root-knot nematode infestations. Pflanzenschtz-Nachrichten 24,