First Report of Korean Cyst Nematode,Heterodera koreana,Parasitic on Bamboo,Phyllostachys nigra,from Iran

Bamboo is grown sporadically in the north of Iran and is confined to very limited areas. The history of growing bamboo was to some extent simultaneous with the entrance, commencement, and growth of the tea industry in the north about a century ago. The bamboo was used for making baskets to transfer the harvested tea foliage from farm to the factory and other linked functions. A main area allocated for bamboo growing is located in Lahidjan Agricultural Research Station (LARS) in the north of Iran, where several species of bamboo were cultivated in an area of 5 ha. The species include five species of Phyllostachys (viz., P. aurea, P. bambusoides, P. decora, P. nigra, P. vivax) and one species of Arundinaria gigantean, Pleioblastus fortune, and Semiarundinaria fastuosa; however, only P. aurea and P. nigra have been precisely identified. A survey on plant parasitic nematodes associated with bamboo mainly on P. nigra in LARS revealed second-stage juveniles of cyst forming nematode in soil samples. Further analysis of root and soil samples led to recovery of a cyst nematode belonging to the genus Heterodera and the Afenestrata group. Cysts, vulval cone, and second-stage juveniles were studied for morphological and morphometric features. The classical identification was followed by amplification of the ribosomal RNA-ITS region and the D2-D3 expansion segments of 28S large-subunit rRNA gene; the amplified fragments were sequenced, edited, and compared with those of the corresponding published gene sequences. New D2-D3 and rRNA-ITS gene sequences were deposited in the GenBank database under the accession numbers {"type":"entrez-nucleotide","attrs":{"text":"KR818910","term_id":"924272655","term_text":"KR818910"}}KR818910 and {"type":"entrez-nucleotide","attrs":{"text":"KR818911","term_id":"924272662","term_text":"KR818911"}}KR818911, respectively. Based on the morphological and molecular data, the species of the cyst-forming nematode was identified as H. koreana (Vovlas et al., 1992; Mundo-Ocampo et al., 2008). The body contour of cysts was mainly subspherical, vey often with irregular shape (Fig. 1A), yellowish to light brown, thin cuticle with fine zigzag pattern, without fenestration, lacking bulla, and underbridge. Vulval lips protruded, cuticular pattern of vulval cone with a tuberculate area (Fig. 2B), and vagina embedded into vulval lips. The second-stage juveniles cylindrical and slender, hemispherical cephalic framework, with three lines in lateral field, well-developed rounded stylet knobs, tail conoid tapring to fine rounded terminus, phasmids posterior to anus. The cyst measurements were (n = 21) length 502 ± 70 (420 to 640) µm; width = 408 ± 60 (320 to 520) µm; length/width = 1.23 ± 0.09 (1.07 to 1.5) µm. The morphometric characters of vulval cone were measured (n = 7): fenestral length = 62.4 ± 6.5 (51 to 71) µm; fenestral width = 50.7 ± 3.2 (45 to 54) µm; vulval slit = 51.9 ± 4.3 (46 to 59) µm; distance from vulva to anus = 51.3 ± 4.4 (43 to 56) µm. Second-stage juveniles showed the following morphometric characters (n = 14): L = 455 ± 11.3 (437 to 472) µm; a = 29.9 ± 0.9 (28.3 to 31.5); b΄ = 2.7 ± 0.4 (2.2 to 3.5); c = 7.4 ± 0.9 (6 to 8.9); ć = 6.1 ± 0.4 (5.1 to 6.7); lip region height = 3 µm; lip region width = 7.5 ± 0.5 (7 to 8) µm; stylet length = 18.1 ± 0.5 (17 to 19) µm; anterior end to median bulb = 72.2 ± 1.7 (70 to 75) µm; anterior end to secretory-excretory pore = 99.7 ± 2.5 (96 to 103) µm; maximum body width = 15.2 ± 0.4 (15 to 16) µm; body width at anus = 10.1 ± 1 (8 to 11) µm; tail length = 62.0 ± 6.9 (51 to 74) µm; hyaline part of tail = 44.0 ± 1.8 (40 to 47) µm. The egg measurements for 11 individuals were length = 102.5 ± 7.9 (93 to 119) µm; width = 39.3 ± 4.2 (33 to 46) µm; length/width = 2.6 ± 0.3 (2.0 to 3.1). The morphology, morphometric characters and molecular data of the population of H. koreana isolated from bamboo in Iran are in agreement with those previously reported for this species (Vovlas et al., 1992; Mundo-Ocampo et al., 2008). At present, five species of Heterodera belonging to the Cyperi and Afenestrata groups were reported from bamboo, H. bamboosi (Kaushal and Swarup, 1988; Wouts and Baldwin, 1998) on Bambusa sp. from India; H. koreana on P. pubescence, P. aurea, and P. nigra from South Korea and the United States; and H. hainanensis (Zhuo et al., 2013), H. fengi (Wang et al., 2013), and H. guangdongensis (Zhuo et al., 2014) on P. pubescence from China; thus showing host suitability of bamboo for at least five species of cyst-forming nematodes. A greenhouse test performed by planting rice seed cv. Hashemi in soil containing H. koreana showed successful multiplication of Korean cyst nematode on rice seedlings after 2 mon. The exact date of the establishment of bamboo plantation in LARS is not precisely clear, but it indicates that the Korean cyst nematode was most likely brought with the imported bamboo seedlings from unknown origin several decades ago. According to our best knowledge, this is the first report of occurrence of H. koreana from Iran. So far the Korean cyst nematode was reported from South Korea, Thailand, and the United States, Florida (from nurseries); this study includes the distribution of this cyst-forming nematode in Iran and expands the information of the occurrence of H. koreana for the world.     

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.     

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.     

Relationship of Resistance to Meloidogyne chitwoodi (race 2) and M. hapla in Alfalfa

In the Pacific Northwest, alfalfa (Medicago sativa) is host to two species of root-knot nematodes, including race 2 of the Columbia root-knot nematode (Meloidogyne chitwoodi) and the northern root-knot nematode (Meloidogyne hapla). In addition to the damage caused to alfalfa itself by M. hapla, alfalfa’s host status to both species leaves large numbers of nematodes available to damage rotation crops, of which potato is the most important. A nematode-resistant alfalfa germplasm release, W12SR2W1, was challenged with both nematode species, to determine the correlation, if any, of resistance to nematode reproduction. Thirty genotypes were screened in replicated tests with M. chitwoodi race 2 or M. hapla, and the reproductive factor (RF) was calculated. The distribution of natural log-transformed RF values was skewed for both nematode species, but more particularly for M. chitwoodi race 2, where more than half the genotypes screened were non-hosts. Approximately 30 percent of genotypes were non-hosts or very poor hosts of M. hapla, but RF values for M. hapla on susceptible genotypes were generally much higher than RF values for genotypes susceptible to M. chitwoodi race 2. The Spearman rank correlation was positive (0.52) and significant (p-value = 0.003), indicating there is some relationship between resistance to these two species of root-knot nematode in alfalfa. However the relationship is not strong enough to suggest genetic loci for resistance are identical, or closely linked. Breeding for resistance or immunity will require screening with each species separately, or with different DNA markers if marker-assisted breeding is pursued. A number of genotypes were identified which are non-hosts to both species. These plants will be intercrossed to develop a non-host germplasm.     

The Effects of Selected Antimetabolites and Antibiotics on Reproduction,Embryonic Development and Hatch of Meloidogyne hapla

Meloidogyne hapla egg-laying was unaffected by a 3-day immersion in 40 ppm concentrations of 6-azauracil, 5-bromodeoxyuridine, or streptomycin sulfate in physiological saline. Comparable exposure to 1-20 ppm cycloheximide irreversibly inhibited egg-laying, but with exposures of 1, 3, or 9 hr, the effect was partly reversible. Of the few eggs laid after the nematodes were transferred to physiological saline, many were abnormally developed. Most of the unlaid eggs extracted from the uteri of cycloheximide-treated nematodes were nonviable. Oogenesis was irreversibly inhibited by the treatment. Cycloheximide stimulated embryonic development and some early hatching, but later hatching was inhibited.     

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.     

Importance of Temperature in the Pathology of Meloidogyne hapla and M. chitwoodi on Legumes

Effects of temperatures on the host-parasite relationships were studied for three legume species and four populations of root-knot nematodes from the western United States. The nematode populations were Meloidogyne hapla from California (MHCA), Utah (MHUT), and Wyoming (MHWY), and a population of M. chitwoodi from Utah (MCUT). The legumes were milkvetch (Astragalus cicer), alfalfa (Medicago sativa), and yellow sweet clover (Melilotus officinalis). All milkvetch plants survived inoculation with all nematode populations, while alfalfa and yellow sweet clover were more susceptible. On yellow sweet clover, MHCA was most pathogenic at 30 °C based on suppression of shoot growth while MHUT, MHWY, and MCUT were most pathogenic at 25 °C. All nematode populations suppressed growth of yellow sweet clover more than growth of milkvetch and alfalfa. The reproductive factor (Rf = final nematode population/initial nematode population) of MHCA was positively correlated (r = 0.83) with temperature between 15 °C and 30 °C. The greatest Rf occurred on alfalfa inoculated with MHCA at 30 °C. The Rf of MHUT, MHWY, and MCUT were positively correlated (r= 0.76, r= 0.78, and r= 0.73, respectively) with temperature between 15 °C and 25 °C. The Rf values of MHUT and MHWY were similar on all species and exceeded the Rf of MCUT at all temperatures (P < 0.05).     

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.     

Entomopathogenic Nematodes and Bacteria Applications for Control of the Pecan Root-Knot Nematode, Meloidogyne partityla, in the Greenhouse

Meloidogyne partityla is a parasite of pecan and walnut. Our objective was to determine interactions between the entomopathogenic nematode-bacterium complex and M. partityla. Specifically, we investigated suppressive effects of Steinernema feltiae (strain SN) and S. riobrave (strain 7–12) applied as infective juveniles and in infected host insects, as well as application of S. feltiae''s bacterial symbiont Xenorhabdus bovienii on M. partityla. In two separate greenhouse trials, the treatments were applied to pecan seedlings that were simultaneously infested with M. partityla eggs; controls received only water and M. partityla eggs. Additionally, all treatment applications were re-applied (without M. partityla eggs) two months later. Four months after initial treatment, plants were assessed for number of galls per root system, number of egg masses per root system, number of eggs per root system, number of eggs per egg mass, number of eggs per gram dry root weight, dry shoot weight, and final population density of M. partityla second-stage juveniles (J2). In the first trial, the number of egg masses per plant was lower in the S. riobrave-infected host treatment than in the control (by approximately 18%). In the second trial, dry root weight was higher in the S. feltiae-infected host treatment than in the control (approximately 80% increase). No other treatment effects were detected. The marginal and inconsistent effects observed in our experiments indicate that the treatments we applied are not sufficient for controlling M. partityla.     

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.     

Root-Knot Nematodes and the Process of Ageing in Plants

Infection of plants by root-knot nematodes is often accompanied by physiological changes characteristic of ageing. Ultra-low tissue luminescence of infected plants indicated oxidation of cell-membrane lipids. Cells with membranes subjected to oxidation lose some of their capacity for water retention. Treating tomato and radish with lidocaine hydrochloride, an inhibitor of lipid oxidation, retarded above-ground symptoms of root-knot nematode infection and of ageing.     

Damage and Management of Meloidogyne hapla Using Oxamyl on Carrot in New York

The northern root-knot nematode (Meloidogyne hapla) is a major pathogen of processing carrot in New York, significantly reducing marketable yield and profitability. Severely infected carrots are stubby, galled and forked and therefore unmarketable. In field microplot trials in 1996 and 1998, the incidence and severity of root-galling increased and the marketable yield of carrot decreased as the initial inoculum density of M. hapla was increased from 0 to 8 eggs/cm³ soil, in mineral or organic soils. The application of oxamyl at planting was effective against M. hapla and its damage to carrots grown in mineral and organic soils. Oxamyl application reduced root-galling severity and increased marketable yield. In commercial fields, the cost-effectiveness of oxamyl application was related to the level of soil infestation with M. hapla.     

Cloning, Sequence, and Expression Analysis of a New MnSOD-Encoding Gene from the Root-Knot Nematode Meloidogyne incognita

A gene encoding a manganese superoxide dismutase (MnSOD) enzyme (Mi-mnsod) was identified and characterized in second-stage juveniles of the root-knot nematode Meloidogyne incognita. The Mi-mnsod gene was found to possess five exons and four introns with (GT/AG) consensus splice-site junctions. The deduced amino acid sequence of Mi-mnsod encodes a putative 25 KDa protein, with conserved amino acid residues of the MnSOD family, including the Parker-Blake signature and four metal-binding sites. The derived amino acid sequence showed high similarity to other eukaryotic MnSODs, including a 23 amino acid N-terminal putative mitochondrial transit peptide. Gene expression was observed throughout the posterior nematode body region with elevated signal intensities at the anterior portion of the intestine. DNA blot analysis and sequencing data showed the occurrence of three putative copies of the MnSOD gene with nucleotide polymorphisms found at the fourth exon and the 3' un-translated region.     

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.     

Reduction of Root-Knot Nematode, Meloidogyne javanica, and Ozone Mass Transfer in Soil Treated with Ozone

Ozone gas (O(3)) is a reactive oxidizing agent with biocidal properties. Because of the current phasing out of methyl bromide, investigations on the use of ozone gas as a soil-fumigant were conducted. Ozone gas was produced at a concentration of 1% in air by a conventional electrical discharge O(3) generator. Two O(3) dosages and three gas flow rates were tested on a sandy loam soil collected from a tomato field that had a resident population of root knot nematodes, Meloidogyne javanica. At dosages equivalent to 50 and 250 kg of O(3)/ha, M. javanica were reduced by 24% and 68%, and free-living nematodes by 19% and 52%, respectively. The reduction for both M. javanica and free-living nematodes was dosage dependent and flow rate independent. The rates of O(3) mass transfer (OMT) through three soils of different texture were greater at low and high moisture levels than at intermediate ones. At any one soil moisture level, the OMT rate varied with soil texture and soil organic matter content. Results suggest that soil texture, moisture, and organic matter content should be considered in determining O(3) dosage needed for effective nematode control.     

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.     

Seasonal Populations of Pratylenchus penetrans and Meloidogyne hapla in Strawberry Roots

Strawberry roots were sampled through the year to determine the populations and distribution of Pratylenchus penetrans and Meloidogyne hapla. Three strawberry root types were sampled—structural roots; feeder roots without secondary tissues; and suberized, black perennial roots. Both lesion and root-knot nematodes primarily infected feeder roots from structural roots or healthy perennial roots. Few nematodes were recovered from soil, diseased roots, or suberized roots. Lesion nematode recovery was correlated with healthy roots. In both 1997 and 1998, P. penetrans populations peaked about day 150 (end of May) and then declined. The decline in numbers corresponded to changes in total strawberry root weight and root type distribution. The loss of nematode habitat resulted from loss of roots due to disease and the transition from structural to suberized perennial roots. Meloidogyne hapla juvenile recovery peaked around 170 days (mid June) in 1997 and at 85, 147, 229, and 308 days (late March, late May, mid August, and early November, respectively) in 1998. There appear to be at least four generations per year of M. hapla in Connecticut. Diagnostic samples from an established strawberry bed may be most reliable and useful when they include feeder roots taken in late May.     

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.     

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.