Cold Hardening of Meloidogyne hapla Second-stage Juveniles

The effect of previous exposure to low temperatures on freezing tolerance was determined for second-stage juveniles of Meloidogyne hapla. Juveniles in 5% polyethylene glycol 20,000 were exposed to 0-24 C for 12-96 hours, and then freezing tolerance was assessed by freezing samples at -4 C for 24 hours, thawing, and determining survival. Freezing tolerance was inversely related to prefreeze temperatures of 4-24 C. Prefreeze exposure to 4 C resulted in fourfold greater freezing tolerance than did exposure to 24 C. Mortality occurred during prefreeze exposure to 0 C. Most of the increase in freezing tolerance at 4 C occurred during the first 12 hours. In soil, prefreeze exposure to 4 C resulted in greater freezing tolerance than did prefreeze exposure to 24 C.     

A PCR Assay to Identify and Distinguish Single Juveniles of Meloidogyne hapla and M. chitwoodi

Random amplified polymorphic DNA (RAPD) bands that distinguish Meloidogyne hapla and M. chitwoodi from each other, and from other root-knot nematode species, were identified using a series of random octamer primers. The species-specific amplified DNA fragments were cloned and sequenced, and then the sequences were used to design 20-mer primer pairs that specifically amplified a DNA fragment from each species. Using the primer pairs, successful amplifications from single juveniles were readily attained. A mixture of four primers in a single PCR reaction mixture was shown to identify single juveniles of M. hapla and M. chitwoodi. To confirm specificity, the primers were used to amplify DNA from several isolates of M. hapla that originated from different crops and locations in North America and also from isolates of M. chitwoodi that differed in host range. In characterizing the M. hapla isolates, it was noted that there was a mitochondrial DNA polymorphism among isolates for cleavage by the restriction endonuclease DraI.     

Hermaphroditism in Meloidogyne hapla

Hermaphrodites were detected in diploid and polyploid isolates of population 86-Va of Meloidogyne hapla. Young hermaphrodites are indistinguishable from normal females. Initially, hermaphrodite ovaries are filled with oocytes at various stages of development. Hermaphroditism is expressed later when young oocytes in the early pachytene region of the growth zone suddenly advance to diakinesis and proceed with maturation divisions, resulting in spermatid production. Spermatogenesis may be initiated shortly after the fourth molt, or later, after a female has produced some eggs. Spermatogenesis may occur in one or both gonads, and it may be initiated in one gonad before the other. Once initiated, spermatogenesis continues for the entire reproductive life of the hermaphrodite. Several thousand spermatozoa accumulate in the ovotestis. Because they do not pass through the oviduct into the spermatotheca, they do not take part in reproduction (nonfunctional hermaphroditism). Among the progeny of hermaphrodites, ca. 50% are hermaphroditic, and the remainder are apparently normal females which, however, produce about 50% hermaphroditic progeny. Two temperature regimes (20-23 C and 27-30 C) did not influence the percentage of hermaphrodites among the progeny. Hermaphroditism could not be transmitted to nonhermaphroditic isolates following attempted crosses between males of hermaphroditic and females of nonhermaphroditic isolates. Although this result suggests cytoplasmic rather than nuclear inheritance, this conclusion is not definitive.     

Influence of Photoperiod and Temperature on Migrations of Meloidogyne Juveniles

Photoperiod influences the migration of M. incognita juveniles toward tomato roots. Approximately 33% migrated vertically 20 cm in 7 days to roots when 12 h dark were alternated with 12 h light. Only 7% migrated when light was constant for 24 h. Vertical migration of M. incognita juveniles was studied at 14, 16, 18, 20, and 22 C. The migration of M. incognita juveniles begins at about 18 C and reaches its maximum at 22 C. The migration of M. hapla and M. incognita juveniles were compared at 14, 18, and 22 C. Juveniles of M. hapla were able to migrate at a lower temperature than those of M. incognita. With M. hapla, there was no significant difference in migration between 18 and 22 C.     

Differential Response of Thor Alfalfa to Meloidogyne chitwoodi Races and M. hapla

Second-stage juveniles (J2) of races 1 and 2 of Meloidogyne chiiwoodi and M. hapla readily penetrated roots of Thor alfalfa and Columbian tomato seedlings; however, few individuals of M. chitwoodi race 1 were able to establish feeding sites and mature on alfalfa. Histopathological studies indicate that J2 of race 1 either failed to initiate feeding sites or they caused cell enlargement without typical cell wall thickening. The protoplasm of these cells coagulated, and juveniles of race 1 did not develop beyond the swollen J2 stage. A few females of race 1 fed on small giant cells and deposited a few eggs at least 20 and 30 days later than M. chitwoodi race 2 and M. hapla, respectively. Failure of race 1 to establish feeding sites was related to egression of J2 from the roots. The M. chitwoodi race 1 J2 egression from alfalfa roots was higher than egression of race 2 and M. hapla. Egression of J2 of M. chitwoodi races 1 and 2 from tomato roots was similar and higher than that of M. hapla. Thus egression plays an important role in the host-parasite relationship of M. chitwoodi and alfalfa.     

Damage Threshold of Meloidogyne hapla to Lettuce in Organic Soil

Lettuce was seeded in pots in the greenhouse and in field microplots in 1991 and 1992. Pots and microplots were filled with untreated or fumigated organic soil infested with Meloidogyne hapla at seven initial population densities (Pi) (0 to 32 eggs/cm³ soil). Lettuce weight, severity of root galling, and number of eggs per root system (Pf) were determined after 8 weeks. At the highest Pi, M. hapla caused yield losses up to 64% in the microplots and plant death in the greenhouse tests. The Seinhorst equation was used to describe the relation between lettuce weight and Pi (r² = 0.73 - 0.98) and to calculate the damage threshold density (T). Values of T were 7 and 8 eggs/cm³ soil in the greenhouse tests of 1991 and 1992, respectively. In the microplot tests, T was 1 egg/cm³ soil in 1991 and 2 eggs/cm³ soil in 1992. The damage threshold was the same in untreated and fumigated soils. At low Pi, root galling was more severe in the pots than in the microplots. Pf increased with increasing Pi of M. hapla in both tests, but declined at Pi > T in the greenhouse tests. The reproduction rate (Pf/Pi) of M. hapla was highest at the lowest Pi.     

Pathogenicity of Two Populations of Meloidogyne hapla Chitwood on Alfalfa and Sainfoin

The pathogenicity of two populations of the northern root-knot nematode, Meloidogyne hapla Chitwood, population 1 (P1) from alfalfa and population 2 (P2) from sainfoin, was studied on both alfalfa and sainfoin for 25 weeks. Alfalfa and sainfoin plants inoculated with P2 had significantly (P ≤ 0.05) higher mortality than plants inoculated with P1. Plant stands over all weeks for the uninoculated control, P1, and P2 were 90.5, 78.5, and 64.0% for alfalfa and 84.5, 51.0, and 41.0% for sainfoin, respectively. The increased virulence of P2 was again shown when means of plant species were combined (inoculation × week of count interaction). Plants inoculated with P2 had significantly higher mortality than either those inoculated with P1 or the uninoculated control beginning at week 7 and continuing through week 25. Plant stands over species at 25 weeks for the uninoculated control, P1, and P2 were 82.5, 29.0, and 18.0%, respectively. Sainfoin was significantly more susceptible to either population than alfalfa (plant species × week of count interaction). Separation between species first occurred after week 7 and continued until week 25. Percentages of plants remaining for alfalfa and sainfoin were 61.5 and 25.0 after 25 weeks. Significantly higher reproduction occurred in the alfalfa plants remaining after 25 weeks in P2 than in P1. Mean number of eggs per root system were 60,371 for P1 and 104,438 for P2, a difference of 42%. The results of this study indicate a need for breeders to adequately sample nematode populations present in the intended area of cultivar use and to design screening procedures to account for population pathogenicity variability.     

Effect of the Mi Gene in Tomato on Reproductive Factors of Meloidogyne chitwoodi and M. hapla

The effect of the Mi gene on the reproductive factor of Meloidogyne chitwoodi and M. hapla, major nematode pests of potato, was measured on nearly isogenic tomato lines differing in presence or absence of the Mi gene. The Mi allele controlled resistance to reproduction of race 1 of M. chitwoodi and to one of two isolates of race 2. No resistance to race 3 of M. chitwoodi or to M. hapla was found. Variability in response to isolates of race 2 may reflect diversity of virulence genotypes heretofore undetected. Resistance to race 1 of M. chitwoodi could be useful in potato if the Mi gene were functional following transferral by gene insertion technology into potato. Since the Mi gene is not superior to RMc₁ derived from Solarium bulbocastanum, the transferral by protoplast fusion appears to offer no advantage.     

Host Tests to Differentiate Meloidogyne chitwoodi Races 1 and 2 and M. hapla

The reproductive factor (R = final egg density at 55 days ÷ 5,000, initial egg density) of Meloidogyne chitwoodi race 2 (alfalfa race) on 46 crop cultivars ranged from 0 to 130. The reproductive efficiency of M. chitwoodi race 1 (non-alfalfa race) and M. chitwoodi race 2 was compared on selected crop cultivars. The basic difference between the two races lay in their differential reproduction on Thor alfalfa and Red Cored Chantenay carrot. M. chitwoodi race 2 reproduced on alfalfa but not on carrot. Conversely, alfalfa was a poor host and carrots were suitable for M. chitwoodi race 1. Based on host responses to M. chitwoodi races and M. hapla, a new differential host test was proposed to distinguish the common root-knot nematode species of the Pacific Northwest.     

Evaluation of Cultivars,Experimental Lines and Plant Introduction Collection of Sainfoin for Resistance to Meloidogyne hapla Chitwood

Stands of several cultivars and experimental lines of sainfoin (Onobrychis viciifolia) were severely reduced (92% average loss) in a field naturally infested with Meloidogyne hapla. Stands of two alfalfa cultivars included in the test were unaffected. In studies conducted in the greenhouse with plants inoculated at the time of seeding, average mortality was 55% for sainfoin entries and 7% for Ladak alfalfa. Little mortality occurred when plants were inoculated after establishment. Three months after inoculation, all sainfoin entries were heavily galled (range of 3.3-3.7 on a scale of 1-4) while roots of Ladak were only slightly galled (rating of 1.6). Intermating of plants selected in the field plots for resistance to M. hapla showed a slight increase in resistance. Of the 147 plant introduction lines tested in the greenhouse, none were resistant to M. hapla.     

Interaction of Vesicular-Arbuscular Mycorrhizae and Cultivars of Alfalfa Susceptible and Resistant to Meloidogyne hapla

The interaction between vesicular-arbuscular mycorrhizal (VAM) fungi and the root-knot nematode (Meloidogyne hapla) was investigated using both nematode-susceptible (Grasslands Wairau) and nematode-resistant (Nevada Synthetic XX) cultivars of alfalfa (Medicago sativa) at four levels of applied phosphate. Mycorrhizal inoculation improved plant growth and reduced nematode numbers and adult development in roots in dually infected cultures of the susceptible cultivar. The tolerance of plants to nematode infection and development when preinfected with mycorrhizal fungi was no greater than when they were inoculated with nematodes and mycorrhizal fungi simultaneously. Growth of plants of the resistant cultivar was unaffected by nematode inoculation but was improved by mycorrhizal inoculation. Numbers of nematode juveniles were lower in the roots of the resistant than of the susceptible cultivar and were further reduced by mycorrhizal inoculation, although no adult nematodes developed in any resistant cultivar treatment. Inoculation of alfalfa with VAM fungi increased the tolerance and resistance of a cultivar susceptible to M. hapla and improved the resistance of a resistant cultivar.     

Control of Pratylenchus penetrans and Meloidogyne hapla and Yield Response of Alfalfa Due to Oxamyl Seed Treatments

Alfalfa (Medicago sativa L. cv. Saranac) seed were soaked for 20 minutes in water, acetone, or methanol containing 10 or 50 mg/ml of oxamyl (Vydate L) or coated with a 2% aqueous cellulose solution containing the same amounts of oxamyl. Seed were analyzed for oxamyl by HPLC immediately after treatment and after 9 and 26 months of storage. Oxamyl content of alfalfa seed did not decline after 26 months of storage. The effects of seed treatment on growth of alfalfa and nematode control were examined using soils infested with Pratylenchus penetrans and Meloidogyne hapla. Germination was not affected by any of the seed treatments. Twenty-one days after sowing, the total growth of alfalfa seedlings grown from seed treated with 50 mg/ml of oxamyl in P. penetrans-infested soils had increased by 62% over controls. Nodulation per pot increased by as much as 267%, and the densities of P. penetrans per gram of root were reduced by as much as 73% compared to control plants. In M. hapla-infested soils, increases in plant growth (32%) and nodulation (71%) also occurred with oxamyl-treated seeds. Root gall reduction (86%) was also substantial due to oxamyl seed treatment.     

Interaction of Ditylenchus dipsaci and Meloidogyne hapla on Resistant and Susceptible Plant Species

Numbers ofDitylenchus dipsaci or Meloidogyne hapla invading Ranger alfalfa, Tender crop bean, Stone Improved tomato, AH-14 sugarbeet, Yellow sweet clover, and Wasatch wheat from single inoculations were not significantly different from numbers by invasion of combined inoculations. D. dipsaci was recovered only from shoot and M. hapla only from root tissue. Combined inoculations did not affect reproduction of either D. dipsaci or M. hapla. D. dipsaci suppressed shoot growth of all species at 15-30 C, and M. hapla suppressed shoot growth of tomato, sugarbeet, and sweet clover at 20, 25, and 30 C. There was a positive correlation (P < 0.05) between shoot and root growth suppression by D. dipsaci on all cultivars except wheat at 20 C and tomato at 30 C. M. hapla suppressed (P < 0.05) root growth of sugarbeet at 20-50 C and wheat at 30 C. Growth suppression was synergistic in combined inoculations of sweet clover shoot growth at 15 C and root growth at 20-30 C, wheat root growth at 15 and 20 C, and tomato root growth at 15-30 C (P < 0.05) D. dipsaci invasions caused mortality of alfalfa and sweet clover at 15-30 C and sugarbeet at 20-30 C. Mortality rates of alfalfa and sweet clover increased synergistically (P < 0.05) from combined inoculations.     

Isolates of Meloidogyne hapla with Distinct Mitochondrial Genomes

Because two conflicting reports of the structure of the Meloidogyne hapla mitochondrial genome exist, we compared the mitochondrial DNA (mtDNA) purified from two isolates of M. hapla: one from San Bernardino County in southern California (BRDO) and the other from England. The authenticity of the BRDO isolate in particular was confirmed by examination of morphological characters, isoenzyme analysis, and differential host range tests. Restriction analysis revealed that mtDNA from the BRDO and English isolates corresponded to only the structure first reported, although significant differences between the two isolates were apparent. Southern blots probed with cloned, cytochrome oxidase I (cox-l) DNA from Romanomermis culicivorax mtDNA confirmed that the analyzed DNA was of mitochondrial origin. Thus, M. hapla has at least two distinct but presumably related mitchondrial genomes, plus at least one very different structure. These data are discussed with reference to recent molecular diagnostic and phylogenetic analyses of Meloidogyne.     

Tolerance to Rotylenchulus reniformis and Resistance to Meloidogyne incognita Race 3 in High-Yielding Breeding Lines of Upland Cotton

Field experiments in 1992 and 1994 were conducted to determine the effect of Rotylenchulus reniformis, reniform nematode, on lint yield and fiber quality of 10 experimental breeding lines of cotton (Gossypium hirsutum) in untreated plots or plots fumigated with 1,3-dichloropropene. Controls were La. RN 1032, a germplasm line possessing some resistance to R. reniformis, and Stoneville 453, a cultivar that is susceptible to reniform nematode. Several breeding lines produced greater lint yields than Stoneville 453 or La. RN 1032 in both fumigated and untreated plots. Average lint yield suppression due to R. reniformis for six of the 10 breeding lines was less than half of the 52% yield reduction sustained by Stoneville 453. In growth chamber experiments, R. reniformis multiplication factors for La. RN 1032 and breeding lines N222-1-91, N320-2-91, and N419-1-91 were significantly lower than on Deltapine 16 and Stoneville 453 at 6 weeks after inoculation. R. reniformis populations increased by more than 50-fold on all entries within 10 weeks. In growth chambers, the breeding lines N220-1-92, N222-1-91, and N320-2-91 were resistant to Meloidoglyne incognita race 3; multiplication factors were ≤1.0 at both 6 weeks and 10 weeks after inoculation compared with 25.8 and 26.5 for Deltapine 16 at 6 and 10 weeks after inoculation, respectively, and 9.1 and 2.6 for Stoneville 453. Thus, the results indicate that significant advances have been made in developing improved cotton germplasm lines with the potential to produce higher yields in soils infested with R. reniformis or M. incogaita. In addition to good yield potential, germplasm lines N222-1-91 and N320-2-91 appear to possess low levels of resistance to R. reniformis and a high level of resistance to M. incognita. This germplasm combines high yield potential with significant levels of resistance to both R. reniformis and M. incognita.     

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.     

Host-Specific Pathogenicity and Genome Differences between Inbred Strains of Meloidogyne hapla

Five isolates of M. hapla originating from the Netherlands and California were inbred by sequential transfer of single egg masses to produce six strains. Cytological examination showed that oocytes of these strains underwent meiosis and had n = 16 chromosomes. Strains were tested for ability to infect and to develop on several hosts by in vitro assays. The two strains from California infected tomato roots at a higher rate than those from the Netherlands, but no difference among strains was seen for ability to develop on tomato with or without the resistance gene Mi-1. All strains developed on the common bean cultivar Kentucky Wonder, but strains differed in ability to develop on the nematode-resistant cultivar NemaSnap. Strain-specific differences were also seen in ability to infect and to develop on Solanum bulbocastanum clone SB-22. Strain VW13, derived from nematodes treated with the mutagen EMS, was defective in ability to infect tomato and potato roots in vitro. Comparison of DNA using AFLP markers showed an average of 4% of the bands were polymorphic across the six strains, but no correlation was observed between the geographical origin or virulence and DNA polymorphism pattern.     

Characterization of Carbohydrates on the Surface of Second-stage Juveniles of Meloidogyne spp.

Fluorescent conjugates of the lectins soybean agglutinin (SBA), Concanavalin A (Con A), wheat germ agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and Limulus polyphemus agglutinin (LPA) bound primarily to amphidial openings and amphidial secretions of viable, preinfective second-stage juveniles (J2) of Meloidogyne incognita races 1 and 3 (Mil, Mi3) and M. javanica (Mj). No substantial difference in fluorescent lectin binding was observed among the populations examined. Binding of only LOT and LPA were inhibited in the presence of 0.1 M competitive sugar. Structural differences in amphidial carbohydrate complexes among populations of Mi 1, Mi3, and Mj were revealed by glycohydrolase treatment of preinfective J2 and subsequent labeling with fluorescent lectins. A quantitative microfiltration enzyme-linked lectin assay revealed previously undetected differences in lectin binding to nonglycohydrolase-treated J2. Freinfective J2 of Mj bound the greatest amount of SBA, LOT, and WGA, whereas J2 of Mil bound the most LPA.     

Host Suitability of Twelve Leguminosae Species to Populations of Meloidogyne hapla and M. chitwoodi

Legumes of the genera Astragalus (milkvetch), Coronilla (crownvetch), Lathyrus (pea vine), Lotus (birdsfoot trefoil), Medicago (alfalfa), Melilotus (clover), Trifolium (clover), and Vicia (common vetch) were inoculated with a population of Melaidogyne chitwoodi from Utah or with one of three M. hapla populations from California, Utah, and Wyoming.Thirty-nine percent to 86% of alfalfa (M. scutellata) and 10% to 55% of red clover (T. pratense) plants survived inoculation with the nematode populations at a greenhouse temperature of 24 ± 3°C. All plants of the other legume species survived all nematode populations, except 4% of the white clover (T. repens) plants inoculated with the California M. hapla population. Entries were usually more susceptible to the M. hapla populations than to M. chitwoodi. Galling of host roots differed between nematode populations and species. Root-galling indices (1 = none, 6 = severely galled) ranged from 1 on pea vine inoculated with the California population of M. hapla to 6 on yellow sweet clover inoculated with the Wyoming population of M. hapla. The nematode reproductive factor (Rf = final nematode population/initial nematode population) ranged from 0 for all nematode populations on pea vine to 35 for the Wyoming population of M. hapla on alfalfa (M. sativa).     

Biological Control of Meloidogyne hapla on Alfalfa and Tomato with the Fungus Meria coniospora

This study was to determine whether Arthrobotrys flagrans, A. oligospora, and Meria coniospora would control the root-knot nematode Meloidogyne hapla on alfalfa and tomato. Alfalfa seeds were coated with a fungus-rye powder in 2% cellulose and were planted in infested soil. Three-week-old seedlings from seed treated with M. coniospora had 60% and 58% fewer galls in two experiments than did seedlings from untreated seeds. Numbers of J2 in the soil were not reduced. Plant growth did not improve. When seed of tomato were coated with M. coniospora and planted in M. hapla-infested soil, roots had 34% fewer galls and 47% fewer J2 in the soil at 28 days. After 56 days there was no reduction in J2 numbers. Plant growth did not improve. When roots of tomato transplants were dusted with M. coniospora fungus-rye powder or sprayed with a spore suspension before planting in M. hapla-infested soil, 42% and 35%, respectively, fewer galls developed in 28 days on treated roots than on roots not treated with fungus. The numbers of J2 extracted from roots or recovered from soil were not reduced, however, and plant growth did not improve.