Infection of Cultured Thin Cell Layer Roots of Lycopersicon esculentum by Meloidogyne incognita

A new aseptic culture system for studying interactions between tomato (Lycopersicon esculentum) and Meloidogyne incognita is described. Epidermal thin cell layer explants from peduncles of tomato produced up to 20 adventitious roots per culture in 4-9 days on Murashige &Scoog medium plus kinetin and indole acetic acid. Rooted cultures were transferred to Gamborg''s B-5 medium and inoculated with infective second-stage juveniles. Gall formation was apparent 5 days after inoculation and egg production by mature females occurred within 25 days at 25 C in the susceptible genotypes Rutgers and Red Alert. Resistant genotypes LA655, LA656, and LA1022 exhibited a characteristic hypersensitive response. This system provides large numbers of cultured root tips for studies on the molecular basis of the host-parasite relationship.     

Reproduction of Virulent Isolates of Meloidogyne incognita on Susceptible and Mi-resistant Tomato

The reproductive potential of natural and laboratory-selected Meloidogyne incognita isolates virulent against the tomato Mi resistance gene, all derived from a single egg-mass, were compared when the nematodes were inoculated on susceptible and resistant tomato. Fewer second-stage juveniles (P = 0.01) of the two virulent populations selected under laboratory conditions matured to females on the resistant tomato compared to the susceptible cultivar. In contrast, no differences were found between the number of egg masses produced on the resistant versus the susceptible tomato by the two natural virulent isolates. No clear general trends concerning the fecundity of the females could be inferred from the comparative analysis of the numbers of eggs per egg mass x tomato cultivar combination. These observations suggested that the genetic changes induced under environmentally controlled nematode growth might be different from those occurring in natural Mi-resistance breaking biotypes grown without environmental control.     

An In Vitro Test for Temperature Sensitivity and Resistance to Meloidogyne incognita in Tomato

An in vitro root explant tissue culture technique is described for determining susceptibility of tomato (Lycopersicon esculentum Mill.) breeding lines and cultivars to the root-knot nematode Meloidogyne incognita. Root explants were taken from 2-day-old seedlings cultured for 30 days at 28 C on Gamborg''s B-5 medium with or without nematode inoculum. The remaining portion of the root and stem from the excised root explants was transferred to soil in pots and grown to maturity in the greenhouse. In vitro root explants were evaluated for growth and occurrence of juveniles, adults, and egg masses. The regenerated plants were used to produce more seed, The proposed technique is simple, reliable, and adapted to routine screening of large numbers of F₁ and F₂ samples, and it utilizes less space than tests performed on intact plants in the greenhouse or growth chamber. Evidence is presented also on the breakdown of resistance to M. incognita under high temperature stress using this in vitro root explant technique.     

Colored Mulches Affect Yield of Fresh-market Tomato Infected with Meloidogyne incognita

The effects of different-colored polyethylene mulches on the quantity and spectra of reflected light, earliness of fruit set, fruit yield and quality, and root-knot disease were studied in field-grown, staked tomato (Lycopersicon esculentum). White mulch reflected more photosynthetic light and a lower far-red-to-red ratio than red mulch, whereas black mulch reflected less than 5 percent of any color. Soil temperatures and fruit yields were recorded for tomato plants inoculated with Meloidogyne incognita race 3 at initial populations of 0, 1,000, 10,000, 50,000, or 100,000 eggs/plant and grown over black, white, or red plastic mulch in both spring and fall. Soil temperatures were lower under white mulch than under red or black mulch. Tomato yields declined as inoculum level increased. Plants grown over red mulch in the spring and inoculated with 50,000 eggs of M. incognita had greater early marketable yields than similarly inoculated plants grown over black or white mulch. Tomato plants inoculated with 100,000 eggs and grown over white mulch or red mulch in the spring had greater total yields per plot than similar plants grown over black mulch (7.39 kg and 7.71 kg vs. 3.65 kg, respectively).     

Survival of Paecilomyces lilacinus in Selected Carriers and Related Effects on Meloidogyne incognita on Tomato

Laboratory and microplot experiments were conducted to determine the influence of carrier and storage of Paecilomyces lilacinus on its survival and related protection of tomato against Meloidogyne incognita. Spores of P. lilacinus were prepared in five formulations: alginate pellets (pellets), diatomaceous earth granules (granules), wheat grain, soil, and soil plus chitin. Fungal viability was high in wheat and granules, intermediate in pellets, and low in soil and chitin-amended soil stored at 25 ± 2 C. In 1985 P. lilacinus in field microplots resulted in about a 25% increase in tomato yield and 25% gall suppression, compared with nematodes alone. Greatest suppression of egg development occurred in plots treated with P. lilacinus in pellets, wheat grain, and granules. In 1986 carryover protection of tomato against M. incognita resulted in about a threefold increase in tomato fruit yield and 25% suppression of gall development, compared with plants treated with nematodes alone. Higher numbers of fungus-infected egg masses occurred in plots treated with pellets (32%) than in those treated with chitin-amended soil (24%), wheat (16%), granules (12%), or soil (7%). Numbers of fungal colony-forming units per gram of soil in plots treated with pellets were 10-fold greater than initial levels estimated at planting time in 1986.     

Optimal Levels of Meloidogyne incognita Inoculum for Infection of Tomato and Peach in Vitro

Penetration of second-stage juveniles (J2) of Meloidogyne incognita into tomato root explants and in vitro propagated peach plantlet roots were compared. Five inoculum levels were used: 25, 50, 75, 100, and 200 J2 for tomato; and 50, 100, 200, 500, and 1,000J2 for peach. The greatest root penetration into tomato was 30% at the 75 J2 level, but the maximum penetration into peach roots was only 8% at the 200 J2 level. The difference (P = 0.05) in penetration of M. incognita at all inoculum levels into these two hosts indicates that penetration versus inoculum density for in vitro studies need to be determined for different plant species.     

Reproduction of Mi-Virulent Meloidogyne incognita Isolates on Lycopersicon spp.

Selection of detectable numbers of Mi-virulent root-knot nematodes has necessitated a greater understanding of nematode responses to new sources of resistance. During the course of this research, we compared the reproduction of four geographically distinct Mi-virulent root-knot nematode isolates on three resistant accessions of Lycopersicon peruvianum. Each accession carried a different resistant gene, Mi-3, Mi-7, or Mi-8. All nematode isolates were verified as Meloidogyne incognita using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of Mi-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the Mi-7 carrying accession and least on the Mi-8 carrying accession. In general, Mi-3 behaved similar to the Mi-8 carrying accession. Reproduction of the four nematode isolates was also compared on both Mi and non-Mi-carrying L. esculentum cultivars and a susceptible L. peruvianum accession. Resistance mediated by Mi in L. esculentum still impacted the Mi-virulent nematodes with fewer eggs per g of root on the resistant cultivar (P ≤ 0.05). Preliminary histological studies suggests that Mi-8 resistance is mediated by a hypersensitive response, similar to Mi.     

Impact of Paecilomyces lilacinus Inoculum Level and Application Time on Control of Meloidogyne incognita on Tomato

Microplot experiments were conducted to evaluate the effects of inoculum level and time of application of Paecilomyces lilacinus on the protection of tomato against MeIoidogyne incognita. The best protection against M. incognita was attained with 10 and 20 g of fungus-infested wheat kernels per microplot which resulted in a threefold and fourfold increase in tomato yield, respectively, compared with tomato plants treated with this nematode alone. Greatest protection against this pathogen was attained when P. lilacinus was delivered into soil 10 days before planting and again at planting. Yield was increased twofold compared with yield in nematode-alone plots and plots with M. incognita plus the fungus. Percentages of P. lilacinus-infected egg masses were greatest in plots treated at midseason or at midseason plus an early application, compared with plots treated with the fungus 10 days before planting and (or) at planting time.     

Meloidogyne incognita Resistance Characteristics in Tomato Genotypes Developed for Processing

Nine resistant processing tomato (Lycopersicon esculentum) cultivars and advanced lines were compared with four susceptible cultivars in 1,3-dichloropropene-fumigated and nontreated plots on Meloidogyne incognita-infested sites over 3 years. Yield of all resistant genotypes grown in nontreated and nematicide-treated plots did not differ and was greater than yield of susceptible genotypes. M. incognita initial soil population densities caused 39.3-56.5% significant (P = 0.05) yield suppressions of susceptible genotypes. Nematode injury to susceptible plants usually caused both fruit soluble solids content and pH to increase significantly (P = 0.05). Only trace nematode reproduction occurred on resistant genotypes in nontreated plots, whereas large population density increases occurred on susceptible genotypes. Slightly greater nematode reproduction occurred on resistant genotypes at the southern desert location, where soil temperature exceeded 30 C, than at other locations. At two locations resistant MOX 3076 supported greater reproduction than other resistant genotypes.     

Histology of the Interactions of Paecilomyces lilacinus with Meloidogyne incognita on Tomato

Excised tomato roots were examined histologically for interactions of the fungus Paecilomyces lilacinus and Meloidogyne incognita race 1. Root galling and giant-cell formation were absent in tomato roots inoculated with nematode eggs infected with P. lilacinus. Few to no galls and no giant-cell formation were found in roots dipped in a spore suspension of P. lilacinus and inoculated with M. incognita. Numerous large galls and giant cells were present in roots inoculated only with M. incognita. P. lilacinus colonized the surface of epidermal cells as well as the internal cells of epidermis and cortex. The possibility of biological protection of plant surfaces with P. lilacinus against root-knot nematodes is discussed.     

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

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

Varietal Response of Tomato to the Interaction of Salinity and Meloidogyne incognita Infection

Response of tomato (Lycopersicon esculentum) cultivars to a range of conductivity levels was tested in the presence and absence of Meloidogyne incognita. The conductivity levels were produced by appropriate adjustment of a 1:1 solution of sodium chloride and calcium chloride. The growth of M. incognita resistant (''Beefmaster'' and ''Atkinson'') and susceptible (''Hunts 2580'' and ''Ronita'') tomato plants was inversely related to soil salinity between EC_e 0 and 5 mmhos/cm. Nematode inoculation of salt-stressed plants significantly reduced plant height, fresh and dry weight, number of flowers, and fruit weight in most cultivars. In Hunts 2580, flower number and fruit weight increased; apparently flower production shifted from determinate to indeterminate, with negative implications for mechanical harvesting. Nematode reproduction on susceptible varieties also decreased with increase in salinity.     

Biomass Partitioning in Tomato Plants Infected with Meloidogyne incognita

Tomato plants were inoculated with Meloidogyne incognita at initial populations (Pi) of 0, 1, 10, 50, 100, and 200 (x 1,000) eggs per plant and maintained in a growth chamber for 40 days. Total fresh biomass (roots + shoots) at harvest was unchanged by nematode inoculation with Pi of 1 x 10⁵ eggs or less. Reductions in fresh shoot weight with increasing Pi coincided with increases in root weight. Total fresh biomass declined with Pi above 1 x 10⁵ eggs, whereas total dry biomass declined at Pi above 1 x 10⁴ eggs. The greatest reduction percentages in fresh shoot biomass induced by root-knot nematodes occurred in the stem tissue, followed by the petiole + rachis; the least weight loss occurred in the leaflets. Although biomass varied among shoot tissues, the relationship between biomass of various shoot tissues and Pi was described by quadratic equations. The linear and quadratic coefficients of the equations (stem, petiole + rachis, or leaflets on Pi) did not differ among tissues when calculations were based on standardized values. Meloidogyne incognita-infected plants had thinner leaves (leaf area/leaf weight) than did uninfected plants. Reductions in leaf weight and leaf area with nematode inoculation occurred at nodes 5-15 and 4, 6-14, respectively. Losses in plant height and mass due to nematodes reflected shorter internodes with less plant mass at each node.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin

Avermectins are macrocyclic lactones produced by Streptomyces avermitilis. Abamectin is a blend of B_1a and B_1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton and some vegetable crops. No LD₅₀ values, data on nematode recovery following brief exposure, or effects of sublethal concentrations on infectivity of the plant-parasitic nematodes Meloidogyne incognita or Rotylenchulus reniformis are available. Using an assay of nematode mobility, LD₅₀ values of 1.56 μg/ml and 32.9 μg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr. Mortality of M. incognita continued to increase following a 1 hr exposure, whereas R. reniformis mortality remained unchanged at 24 hr after the nematodes were removed from the abamectin solution. Sublethal concentrations of 1.56 to 0.39 μg/ml for M. incognita and 32.9 to 8.2 μg/ml for R. reniformis reduced infectivity of each nematode on tomato roots. The toxicity of abamectin to these nematodes was comparable to that of aldicarb.     

Effects of Population Densities of Meloidogyne hapla on Growth and Yield of Tomato

Growth and yield of ''Veebrite'' tomato were studied in 20-cm (i.d.) clay-tile microplots containing initially 260, 1,840, 6,120, or 27,950 Meloidogyne hapla larvae/kg of soil. Low nematode numbers stimulated, and the highest nematode population suppressed, vegetative plant growth. More tomatoes, with a higher total weight, were harvested from plants infested with 260 and 1,840 nematode larvae at planting than from those with initial densities of 6,120 and 27,950 larvae. At the two highest densities, the cumulative fruit production (weight) was suppressed by 10% and 40%, respectively. The increase in growth and yield at the lower densities appeared to be due to an increase in the size of the root systent. However, at the higher densities, yield was no longer directly related to root weight. The reproduction factor of M. hapla was negatively correlated with initial density; for the lowest and highest initial densities, it was 96X and 7X at midseason, and 354X and 3X at harvest, respectively. The equilibrium density was 63,000 larvae/kg of soil; initial densities larger than 2,000 larvae/kg of soil may require control.     

Characterization of Anionic Peroxidases in Tomato Isolines Infected by Meloidogyne incognita

Changes in peroxidase activity during nematode infection were studied using root extracts of tomato near-isogenic lines differing in resistance to Meloidogyne incognita. Total peroxidase activity increased slightly in crude extracts of four susceptible isolines but doubled in two resistant lines, Monita and Motaci. Nematode infection enhanced levels of both p-phenylenediamine-pyrocatechol oxidase and syringaldazine oxidase 7 days after inoculation, especially in resistant lines. This elevated peroxidase activity in resistant isolines was caused by an increase in anionic peroxidase activity. These enzymes, which likely are involved in lignification, were isolated and purified from tomato isolines by ammonium sulfate precipitation, high performance ion-exchange chromatography, and gel electrophoresis. The purified anionic peroxidase extracts contained an electrophoretic band with Rf 0.51 that was present in extracts of infected but not uninfected roots.     

Resistance in Lycopersicon peruvianum to Isolates of Mi Gene-Compatible Meloidogyne Populations

Root-knot nematode resistance of F₁ progeny of an intraspecific hybrid (Lycopersicon peruvianum var. glandulosum Acc. No. 126443 x L. peruvianum Acc. No. 270435), L. esculentum cv. Piersol (possessing resistance gene Mi), and L. esculentum cv. St. Pierre (susceptible) was compared. Resistance to 1) isolates of two Meloidogyne incognita populations artificially selected for parasitism on tomato plants possessing the Mi gene, 2) the wild type parent populations, 3) four naturally occurring resistance (Mi gene)-breaking populations of M. incognita, M. arenaria, and two undesignated Meloidogyne spp., and 4) a population of M. hapla was indexed by numbers of egg masses produced on root systems in a greenhouse experiment. Artificially selected M. incognita isolates reproduced abundantly on Piersol, but not (P = 0.01) on resistant F₁ hybrids. Thus, the gene(s) for resistance in the F₁ hybrid differs from the Mi gene in Piersol. Four naturally occurring resistance-breaking populations reproduced extensively on Piersol and on the F₁ hybrid, demonstrating ability to circumvent both types of resistance. Meloidogyne hapla reproduced on F₁ hybrid plants, but at significantly (P = 0.01) lower levels than on Piersol.     

Induction of Isoperoxidases in Resistant and Susceptible Tomato Cultivars by Meloidogyne incognita

Isoperoxidases were detected in resistant Rossol and susceptible Roma VF tomato roots uninfected and infected by Meloidogyne incognita. Syringaldazine, guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), and indoleacetic acid (IAA) were used as substrates, and the corresponding peroxidative activities were detected either in cytoplasmic or in cell wall fractions, except for IAA oxidase, which was measured in soluble and microsomal fractions. Isoperoxidase activities and cellular locations were induced differently in resistant and susceptible cultivars by nematodes. Nematode infestation markedly enhanced syringaldazine oxidase activity in cell walls of the resistant cultivar. This isoperoxidase is involved in the last step of lignin deposition in plants. Conversely, the susceptible cultivar reacted to M. incognita infection with an increase in cytoplasmic PPD-PC oxidase activity, which presumedly is involved in ethylene production; no changes in cell wall isoperoxidases were observed. IAA oxidase was inhibited in susceptible plants after nematode inoculation, whereas in resistant plants this activity increased in the soluble fraction and decreased in the microsomal fraction.     

Effect of Temperature on Attachment,Development, and Interactions of Pasteuria penetrans on Meloidogyne incognita

The effect of temperature (10, 20, 25, 30, and 35 C) on attachment and development of Pasteuria penetrans on Meloidogyne arenaria race 1 was elevated in growth chambers. The greatest attachment rate of endospores of P. penetrans occurred on second-stage juveniles at 30 C. The bacterium developed more quickly within its host at 30 and 35 C than at 25 C or below. The development of the bacterium within the nematode female was divided into nine recognizable life stages, which ranged from early vegetative thalli to mature sporangia. Mature sporangium was the predominant life stage observed after 35, 40, 81, and 116 days at 35, 30, 25, and 20 C, respectively. The body width and length of M. arenaria females infected with P. penetrans were smaller initially than the same dimensions in uninfected females, but became considerably larger over time at 25, 30, and 35 C. This isolate of P. penetrans also parasitized and completed its life cycle in males of M. arenaria.     

Embryogenesis and Postinfection Development of Meloidogyne konaensis

The effects of temperature on embryogenesis and postinfection development in Meloidogyne konaensis were examined. Embryogenesis was evaluated at 5, 8, 10, 13, 16, 20, 22, 24, 26, 28, 30, 35, and 40 C. No embryonic development occurred at 5 C. Some development, although incomplete, occurred at 8 and 10 C. The rate of embryogenesis was linear from 13 to 30 C, but decreased at 35 C. The lowest egg mortality occurred at 24 C, whereas all eggs died within 24 hours at 40 C. Postinfection development was determined on coffee and tomato in a greenhouse at an average temperature of 30 C and in a growth chamber with a constant temperature of 26 C. Development of M. konaensis J2 to mature female required 38 and 48 days on coffee at 30 and 26 C, respectively. This process took 20 and 26 days, respectively, on tomato.