Reproduction of Meloidogyne javanica on Plant Roots Genetically Transformed by Agrobacterium rhizogenes

Reproduction of Meloidogyne javanica was compared on several Agrobacterium rhizogenes-transformed root cultures under monoxenic conditions. M. javanica reproduced on all transformed roots tested; however, more females and eggs were obtained on potato and South Australian Early Dwarf Red tomato than on bindweed, Tropic tomato, lima bean, or carrot. Roots that grew at moderate rates into the agar and produced many secondary roots supported the highest reproduction. Numbers of females produced in cultures of transformed potato roots increased with increasing nematode inoculum levels, whether inoculum was dispersed eggs or juveniles. Females appeared smaller, produced fewer eggs, and were found in coalesced galls at the higher inoculum levels. The ratio between the final and initial population decreased sharply as the juvenile inoculum increased. The second-stage juvenile was preferred to dispersed eggs or egg masses for inoculation of tissue culture systems because quantity and viability of inoculum were easily assessed. Meloidogyne javanica reared on transformed root cultures were able to complete their life cycles on new transformed root cultures or greenhouse tomato plants.     

Effects of Acibenzolar-S-Methyl Application to Rotylenchulus reniformis and Meloidogyne javanica

Effects of acibenzolar-s-methyl, an inducer of systemic acquired resistance in plants, on Rotylenchulus reniformis and Meloidogyne javanica in vitro and in vivo were determined. A single foliar application of acibenzolar at 50 mg/liter (5 ml of solution per plant) to 7-day-old cowpea or soybean seedlings decreased R. reniformis and M. javanica egg production by 50% 30 days after inoculation. The mechanism of acibenzolar on plant-parasitic nematodes was then investigated. Acibenzolar at 50 to 200 mg/liter did not affect movement of R. reniformis and M. javanica or penetration of second-stage juveniles (J2) of M. javanica on cowpea. However, M. javanica development was slowed and fecundity was reduced in plants treated with acibenzolar. On average, 50% of J2 that penetrated acibenzolar-treated cowpeas developed into mature females with eggs, whereas the other 50% exhibited arrested development. The number of eggs per egg mass was 450 in water-treated cowpeas, whereas the number declined to 250 in acibenzolar-treated plants. Acibenzolar may be responsible for stimulating the plants to express some resistance to the nematodes.     

Effects of Etomopathiogenic Nematodes on Meloidogyne javanica on Tomatoes and Soybeans

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

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

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

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.     

Control of Heterodera carotae,Ditylenchus dipsaci,and Meloidogyne javanica with Fumigant and Nonfumigant Nematicides

Five field trials were conducted in Italy in 1983 and 1984 to test the efficacy of isazofos and benfuracarb in controlling Heterodera carotae on carrot, Ditylenchus dipsaci on onion, and Meloidogyne javanica on tomato. Methyl isothiocyanate (MIT) was tested against H. carotae and M. javanica. Single (10 kg a.i./ha) and split (5 + 5 kg a.i./ha) applications of isazofos gave yield increases of carrot and onion similar to those obtained with DD (300 liters/ha) and aldicarb (10 kg a.i./ha). Population densities of H. carotae in carrot roots at harvest and of M. javanica in tomato roots 2 months after transplanting were also suppressed by isazofos. Benfuracarb (10 kg a.i./ha increased onion yields in a field infested with D. dipsaci, but it was not effective against H. carotae or M. javanica. The efficacy of MIT at 400 and 600 liters/ha was similar to that of MIT + DD (Di-Trapex) at 300 liters/ha. Both nematicides inhibited hatch of H. carotae eggs and decreased the soil population density of M. javanica.     

Detection and Investigation of Soil Biological Activity against Meloidogyne incognita

Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm³ soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm³ soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.     

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.     

Yield Response and Injury Levels of Meloidogyne incognita and M. javanica on the Susceptible Tobacco 'McNair 944'

The effects of Meloidogyne incognita and M. javanica on a susceptible tobacco (Nicotiana tabacum L.) cv. McNair 944 were investigated in field microplots during 1978 and 1979. Three initial inoculum levels—4, 16, and 64 nematode eggs and/or second-stage larvae per 100 cm³ of soil—were used for each nematode species. Data obtained from the experiments included plant yield and the amount of reproduction of the two nematode species. At comparative inoculum levels, M. javanica was more aggressive than M. incognita on tobacco and caused approximately twofold more yield suppression than M. incognita. The calculated initial population of M. incognita, derived from the average for 2 yr, which produced a 7% suppression in plant yield was four eggs and/or second-stage larvae per 100 cm³ of soil; whereas less than one M. javanica egg and/or second-stage larvae per 100 cm³ of soil was needed to achieve similar suppression. Nematode reproduction varied in the 1978 and 1979 tests, but similar trends were observed. Early season M. javanica populations were greater than those of M. incognita, but late season populations of M. incognita were twice anti three times those of M. javanica.     

Comparative Studies on Root Invasion,Root Galling,and Fecundity of Three Meloidogyne spp. on a Susceptible Tobacco Cultivar

Root invasion, root galling, and fecundity of Meloidogyne javanica, M. arenaria, and M. incognita on tobacco was compared in greenhouse and controlled environment experiments. Significantly more M. javanica than M. arenaria or M. incognita larvae were found in tobacco roots at 2, 4, and 6 d after inoculation. Eight days after inoculation there were significantly more M. arenaria and M. javanica than M. incognita larvae. Ten days after inoculation no significant differences were found among the three Meloidogyne species inside the roots. Galls induced by a single larva or several larvae of M. javanica were significantly larger than galls induced by M. incognita: M. arenaria galls were intermediate in size. Only slight differences in numbers of egg masses or numbers of eggs produced by the three Meloidogyne species were observed up to 35 d after inoculation.     

Suitability of Zucchini and Cucumber Genotypes to Populations of Meloidogyne arenaria,M. incognita,and M. javanica

The host suitability of five zucchini and three cucumber genotypes to Meloidogyne incognita (MiPM26) and M. javanica (Mj05) was determined in pot experiments in a greenhouse. The number of egg masses (EM) did not differ among the genotypes of zucchini or cucumber, but the eggs/plant and reproduction factor (Rf) did slightly. M. incognita MiPM26 showed lower EM, eggs/plant, and Rf than M. javanica Mj05. Examination of the zucchini galls for nematode postinfection development revealed unsuitable conditions for M. incognita MiPM26 as only 22% of the females produced EM compared to 95% of the M. javanica females. As far as cucumber was concerned, 86% of the M. incognita and 99% of the M. javanica females produced EM, respectively. In a second type of experiments, several populations of M. arenaria, M. incognita, and M. javanica were tested on zucchini cv. Amalthee and cucumber cv. Dasher II to assess the parasitic variation among species and populations of Meloidogyne. A greater parasitic variation was observed in zucchini than cucumber. Zucchini responded as a poor host for M. incognita MiPM26, MiAL09, and MiAL48, but as a good host for MiAL10 and MiAL15. Intraspecific variation was not observed among the M. javanica or M. arenaria populations. Cucumber was a good host for all the tested populations. Overall, both cucurbits were suitable hosts for Meloidogyne but zucchini was a poorer host than the cucumber.     

Interaction of Meloidogyne javanica with Different Races of Meloidogyne incognita

The interspecific interactions of Meloidogyne javanica with races 1, 2, 3, and 4 of M. incognita on tomato were determined. Impacts of the interactions on fecundity and morphometrics of females were also examined. Mutually inhibitory interactions occurred between M. javanica and the races of M. incognita, but the negative interactions did not reflect in plant growth. Numbers of root galls, egg masses, mature females, total population, fecundity, and reproduction factor declined in concomitant treatments, but the morphometrics of the females remained unaltered. In general, mutual suppressive effects in all parameters were smaller for M. javanica than M. incognita, but some variations occurred among the races of M. incognita. Race 2 appeared to be more competitive than other races. The interaction between the species was not intense; therefore, the species coexist in mixed populations in agricultural fields.     

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

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

Control of Meloidogyne javanica by Formulations of Inula viscosa Leaf Extracts

Inula viscosa is a perennial plant that is widely distributed in Mediterranean countries. Formulations of I. viscosa extracts were tested for their effectiveness in control of Meloidogyne javanica in laboratory, growth chamber, microplot, and field experiments. Oily pastes were obtained by extraction of dry leaves with a mixture of acetone and n-hexane or n-hexane alone, followed by evaporation of the solvents. Emulsifiable concentrate formulations of the pastes killed M. javanica juveniles in sand at a concentration of 0.01% (paste, w/w) or greater and reduced the galling index of cucumber seedlings as well as the galling index and numbers of nematode eggs on tomato plants in growth chamber experiments. In microplot experiments, the hexane-extract formulation at 26 g paste/m² reduced nematode infection on tomato plants in one of two experiments. In a field experiment, a reduction of 40% in root galling index by one of two formulations was observed on lettuce plants. The plant extracts have potential as a natural nematicide, although the formulations need improvement.     

Efficacy of Ethoprop on Meloidogyne hapla and M. chitwoodi and Enhanced Biodegradation in Soil

Responses of egg masses, free eggs, and second-stage juveniles (J2) ofMeloidogyne hapla and M. chitwoodi to ethoprop were evaluated. The results indicated that J2 were the most sensitive, followed by free eggs and egg masses. In general, M. chitwoodi was more susceptible to ethoprop than M. hapla. Ethoprop at 7.2 μg a.i./g soil protected tomato roots from upward migrating M. chitwoodi for 5 weeks. The zone of protection was extended to 10 and 20 cm below the root zone when 3.6 and 7.2 cm water were applied over 8 days. Ethoprop at 1.8, 3.6, and 7.2 μg a.i./g soil degraded faster and killed fewer M. chitwoodi J2 in potato field soil previously exposed to ethoprop than in unexposed soil or sterilized exposed soil. The enhanced biodegradation property of the exposed soil lasted 17 months after the last application of ethoprop. The limited downward movement of ethoprop in the soil, migration of M. chitwoodi J2 into the treated zone, presence of resistant life stage(s) at the time of application, and loss of efficacy due to enhanced biodegradation may have a significant effect on the performance of ethoprop.     

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.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Extraction of Root-associated Meloidogyne incognita and Rotylenchulus reniformis

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

Effects of Inducers of Systemic Acquired Resistance on Reproduction of Meloidogyne javanica and Rotylenchulus reniformis in Pineapple

The potency of the inducers of systemic acquired resistance (SAR), acibenzolar-s-methyl, DL-α-amino-n-butyric acid (AABA), DL-β-amino-n-butyric acid (BABA), γ-amino-n-butyric acid (GABA), p-aminobenzoic acid (PABA), riboflavin, and salicylic acid (SA), in reducing reproduction of Meloidogyne javanica and Rotylenchulus reniformis in pineapple was investigated. All inducers were applied as foliar sprays to 1-mon-old pineapple plants (20 ml/plant) grown in 22-cm-diam. pots in the greenhouse. Two days after application, 10,000 eggs of M. javanica or R. reniformis were inoculated onto the plants. Six months after inoculation, nematode reproduction was measured. Acibenzolar decreased R. reniformis egg production by 58% compared to the nontreated control (P ≤ 0.05). Acibenzolar, BABA, and riboflavin reduced M. javanica egg production by 60% to 64% compared to the nontreated control (P ≤ 0.05). The point in the pineapple SAR pathway that each compound activates may explain the differing results between M. javanica and its giant cells and R. reniformis and its syncytia. Foliar application of acibenzolar at 100 and 200 mg/liter decreased by 30% and 60%, respectively, the number of M. javanica eggs as compared to the nontreated control. Fresh shoot weight of pineapple treated with 50, 100, 200, and 400 mg/liter acibenzolar was reduced by 1.2%, 3.3%, 9.9%, and 33% compared to the nontreated pineapple, respectively (P ≤ 0.05). Foliar application of acibenzolar may activate intrinsic resistance of pineapple to M. javanica and R. reniformis and may have a role in the sustainable management of nematodes in pineapple.     

Enzymatic Digestion of Roots for Recovery of Root-knot Nematode Developmental Stages

Developmental stages of Meloidogyne javanica were successfully released from roots by treatment with commercially available cellulase and pectinase. The average percentage recovery of nematode developmental stages from Dolichos lablab, Elymus glaucus, and Lycopersicon esculentum were as follows: eggs = 526%, J2 = 272%, J3 = 783%, J4 = 549%, adult females = 285%, and total = 425%, expressed as percentages of the counts obtained from stained roots spread on glass plates. Root digestion was more accurate and sensitive in detecting low numbers of nematodes in roots than was the glass plate method. No simple linear, quadratic, or cubic relationship was found between the two methods that would allow a conversion factor to be developed.