Molecular investigation of proteinase inhibitor (PI) gene in tomato plants induced by Meloidogyne species.

BACKGROUND: The plant parasitic nematode genus Meloidogyne parasitize almost all flowering crops. Plants respond with a variety of morphological and molecular mechanisms to reduce the effects of pathogens. Proteinase inhibitors (PI), a special group of plant proteins which are small proteins, involve in protective role in the plants attacked by microorganisms. Still, the plant response using PI against nematodes has not been well understood. Therefore, this study was aimed to determine the expression of proteinase inhibitor I (PI-I) gene subsequent the infection of M. incognita, M. javanica, and M. chitwoodi in tomato plants post nematode infections. Molecular methods were used to determine the PI gene expressions at different days post nematode infections in host tissues. RESULTS: Results revealed that the population of M. incognita species reached the highest level of nematode population followed by M. javanica and M. chitwoodi, respectively. All Meloidogyne species induced expression of PI-I gene reached at the utmost level at 3 days post infection (dpi) in host tissues. Relative gene expression level was sharply dropped at 7 dpi, 14 dpi, and 21 dpi in M. incognita induced gene expression in host tissues. Similar results were observed in host tissues after infection of M. javanica and M. chitwoodi. CONCLUSIONS: The commonalities of plant response across a diverse Meloidogyne species interaction and the expression of PI gene may be related to plant defense system. Increased level of PI gene expressions in early infection days in host tissues induced by parasitic nematodes may share resemblances to the mechanisms of resistance on biotrophic interactions.

Shifts in the Active Rhizobiome Paralleling Low Meloidogyne chitwoodi Densities in Fields Under Prolonged Organic Soil Management.

Plants manipulate their rhizosphere community in a species and even a plant life stage-dependent manner. In essence plants select, promote and (de)activate directly the local bacterial and fungal community, and indirectly representatives of the next trophic level, protists and nematodes. By doing so, plants enlarge the pool of bioavailable nutrients and maximize local disease suppressiveness within the boundaries set by the nature of the local microbial community. MiSeq sequencing of specific variable regions of the 16S or 18S ribosomal DNA (rDNA) is widely used to map microbial shifts. As current RNA extraction procedures are time-consuming and expensive, the rRNA-based characterization of the active microbial community is taken along less frequently. Recently, we developed a relatively fast and affordable protocol for the simultaneous extraction of rDNA and rRNA from soil. Here, we investigated the long-term impact of three type of soil management, two conventional and an organic regime, on soil biota in fields naturally infested with the Columbian root-knot nematode Meloidogyne chitwoodi with pea (Pisum sativum) as the main crop. For all soil samples, large differences were observed between resident (rDNA) and active (rRNA) microbial communities. Among the four organismal group under investigation, the bacterial community was most affected by the main crop, and unweighted and weighted UniFrac analyses (explaining respectively 16.4% and 51.3% of the observed variation) pointed at a quantitative rather than a qualitative shift. LEfSe analyses were employed for each of the four organismal groups to taxonomically pinpoint the effects of soil management. Concentrating on the bacterial community in the pea rhizosphere, organic soil management resulted in a remarkable activation of members of the Burkholderiaceae, Enterobacteriaceae, and Pseudomonadaceae. Prolonged organic soil management was also accompanied by significantly higher densities of bacterivorous nematodes, whereas levels of M. chitwoodi had dropped drastically. Though present and active in the fields under investigation Orbiliaceae, a family harboring numerous nematophagous fungi, was not associated with the M. chitwoodi decline. A closer look revealed that a local accumulation and activation of Pseudomonas, a genus that includes a number of nematode-suppressive species, paralleled the lower M. chitwoodi densities. This study underlines the relevance of taking along both resident and active fractions of multiple organismal groups while mapping the impact of e.g. crops and soil management regimes.

Bacterial Microbiome and Nematode Occurrence in Different Potato Agricultural Soils.

Pratylenchus neglectus and Meloidogyne chitwoodi are the main plant-parasitic nematodes in potato crops of the San Luis Valley, Colorado. Bacterial microbiome (16S rRNA copies per gram of soil) and nematode communities (nematodes per 200 g of soil) from five different potato farms were analyzed to determine negative and positive correlations between any bacterial genus and P. neglectus and M. chitwoodi. Farms showed differences in bacterial communities, percentage of bacterivorous and fungivorous nematodes, and numbers of P. neglectus and M. chitwoodi. The farm with the lowest population of P. neglectus and M. chitwoodi had higher abundances of the bacterial genera Bacillus spp., Arthrobacter spp., and Lysobacter spp., and the soil nematode community was composed of more than 30% of fungivorous nematodes. In contrast, the farm with higher numbers of P. neglectus and M. chitwoodi had a lower abundance of the abovementioned bacterial genera, higher abundance of Burkholderia spp., and less than 25% of fungivorous nematodes. The α-Proteobacteria Rhodoplanes, Phenylobacterium, and Kaistobacter positively correlated with M. chitwoodi, and the Bacteroidia and γ-Proteobacteria positively correlated with P. neglectus. Our results, based largely on co-occurrence analyses, suggest that the abundance of Bacillus spp., Arthrobacter spp., and Lysobacter spp. in Colorado potato soils is negatively correlated with P. neglectus and M. chitwoodi abundance. Further studies will isolate and identify bacterial strains of these genera, and evaluate their nematode-antagonistic activity.

Site-Specific Management of Meloidogyne chitwoodi in Idaho Potatoes Using 1,3-Dichloropropene; Approach, Experiences, and Economics.

Fumigation for nematode management in irrigated potato production systems of Idaho is widely practiced. Soil injection is the only labeled application method for 1,3-dichloropropene that is conventionally applied on a whole-field basis. Plant-parasitic nematode species exhibit spatially variable population densities that provide an opportunity to practice site-specific fumigation to reduce chemical usage and production costs. During 2002 to 2008, 62 fields intended for commercial potato production in eastern Idaho were sampled using a geo-referenced grid sampling system for plant-parasitic nematode population densities. In total, 4,030 grid samples were collected representing nearly 3,200 ha of commercial potato production. Collectively, 73% of the grid samples had Columbia root knot (CRN) (Meloidogyne chitwoodi) population densities below detectable levels. Site-specific fumigation is the practice of varying application rate of a fumigant based on nematode population density. In 2007, 640 ha of potato production were site-specific fumigated for CRN nematode control in eastern Idaho. On average, this practice resulted in a 30% reduction in chemical usage and production cost savings of $209/ha when 1,3-dichloropropene was used as the sole source of nematode suppression. Reductions in usage of 1,3-dichloropropene can exceed 50% if used in combination with a nonfumigant nematicide such as oxamyl. This combination approach can have production cost savings exceeding $200/ha. Based on farm-gate receipts and USDA inspections provided by potato producers from 2001 to 2011, potato tuber yield and quality have not been adversely affected using site-specific fumigation.

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.

Nematodes in dryland field crops in the semiarid pacific northwest United States.

Soils and roots of field crops in low-rainfall regions of the Pacific Northwest were surveyed for populations of plantparasitic and non-plant-parasitic nematodes. Lesion nematodes (Pratylenchus species) were recovered from 123 of 130 non-irrigated and 18 of 18 irrigated fields. Pratylenchus neglectus was more prevalent than P. thornei, but mixed populations were common. Population densities in soil were affected by crop frequency and rotation but not by tillage or soil type (P < 0.05). Many fields (25%) cropped more frequently than 2 of 4 years had potentially damaging populations of lesion nematodes. Pratylenchus neglectus density in winter wheat roots was inversely correlated with grain yield (r(2) = 0.64, P = 0.002), providing the first field-derived evidence that Pratylenchus is economically important in Pacific Northwest dryland field crops. Stunt nematodes (Tylenchorhynchus clarus and Geocenamus brevidens) were detected in 35% of fields and were occasionally present in high numbers. Few fields were infested with pin (Paratylenchus species) and root-knot (Meloidogyne naasi and M. chitwoodi) nematodes. Nematodes detected previously but not during this survey included cereal cyst (Heterodera avenae), dagger (Xiphinema species), and root-gall (Subanguina radicicola) nematodes.

Genetic Analysis of Resistance to Meloidogyne chitwoodi Introgressed from Solanum hougasii into Cultivated Potato.

An accession of Solanum hougasii, a wild tuber-bearing potato species native to Mexico, was found to be resistant to races 1 and 2 of Meloidogyne chitwoodi. A resistant selection was selfed and its progeny possessed the same combined resistance uniformly. A selected resistant seedling from the selfed progeny was crossed to cultivated tetraploid potato (S. tuberosum) to form an F hybrid, and was backcrossed to cultivated tetraploid potato to form a BC population in which resistance to the two races segregated. Progeny of the BC were tested in inoculation experiments with four replicates for each progeny genotype for each race of nematode. Resistance was evaluated on the basis of extracted egg counts from the entire root system of pot-grown plants. Considering resistance to each race separately, for race 1, non-host (Rf

A Pathotype System to Describe Intraspecific Variation in Pathogenicity of Meloidogyne chitwoodi.

Tests of eight Dutch Meloidogyne chitwoodi isolates to the differential set for host races 1 and 2 in M. chitwoodi provided no evidence for the existence of host race 2 in the Netherlands. The data showed deviations from expected reactions on the differential hosts, which raised doubts of the usefulness of the host race classification in M. chitwoodi. The term ''pathotype'' is proposed for groups of isolates of one Meloidogyne sp. that exhibit the same level of pathogenicity on genotypes of one host species. We recommend that the pathotype classification be applied in pathogen-host relationships when several genotypes of a Meloidogyne sp. are tested on several genotypes of one host species. Three pathotypes of M. chitwoodi were identified on Solanum bulbocastanum, suggesting at least two different genetic factors for virulence and resistance in the pathogen and the host species, respectively. The occurrence of several virulence factors in M. chitwoodi will complicate the successful application of resistance factors from S. bulbocastanum for developing resistant potato cultivars.

Survey of crop losses in response to phytoparasitic nematodes in the United States for 1994.

Previous reports of crop losses to plant-parasitic nematodes have relied on published results of survey data based on certain commodities, including tobacco, peanuts, cotton, and soybean. Reports on crop-loss assessment by land-grant universities and many commodity groups generally are no longer available, with the exception of the University of Georgia, the Beltwide Cotton Conference, and selected groups concerned with soybean. The Society of Nematologists Extension Committee contacted extension personnel in 49 U.S. states for information on estimated crop losses caused by plant-parasitic nematodes in major crops for the year 1994. Included in this paper are survey results from 35 states on various crops including corn, cotton, soybean, peanut, wheat, rice, sugarcane, sorghum, tobacco, numerous vegetable crops, fruit and nut crops, and golf greens. The data are reported systematically by state and include the estimated loss, hectarage of production, source of information, nematode species or taxon when available, and crop value. The major genera of phytoparasitic nematodes reported to cause crop losses were Heterodera, Hoplolaimus, Meloidogyne, Pratylenchus, Rotylenchulus, and Xiphinema.

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.

Resistance of Auto- and Allotetraploid Triticeae Species and Accessions to Meloidogyne chitwoodi based on Genome Composition.

The Columbia root-knot nematode Meloidogyne chitwoodi parasitizes several plant species, including grasses that have been developed for semiarid environments, and substantially reduces the productivity of cereals and the longevity of perennial grasses growing under semiarid conditions throughout the intermountain region. Thirty-two auto- and allotetraploid (2n = 28) taxa in the perennial Triticeae were evaluated as possible sources of resistance to M. chitwoodi. Low levels of root galling were observed on roots of all accessions; root-gall indices ranged from 0 (no galls) to 1.95 in the grasses compared to 4.67 for the susceptible 'Ranger' alfalfa check on a scale of 1 to 6. Even though the gall ratings were low, significant (P < 0.01) differences among accessions of the same species, among species, and among genera with different genomes were observed. Within the reproductive indices, which ranged from 0.01 to 1.20 in the grasses compared to 65.38 for the alfalfa check, there was no difference among genera with different genomes and accessions within the same species and genome; however, there was a significant (P < 0.05) difference among species with the same genomes. This variation can be traced to Thinopyrum nodosum (Jaaska-19), which was the only accession with a reproductive factor greater than 1.00. Based on the data, all auto- and allotetraploids are considered resistant to M. chitwoodi.

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 degrees C based on suppression of shoot growth while MHUT, MHWY, and MCUT were most pathogenic at 25 degrees 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 degrees C and 30 degrees C. The greatest Rf occurred on alfalfa inoculated with MHCA at 30 degrees 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 degrees C and 25 degrees 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).

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 RMc derived from Solarium bulbocastanum, the transferral by protoplast fusion appears to offer no advantage.

Reproduction of Meloidogyne chitwoodi on Popcorn Cultivars.

Popcorn cultivars were evaluated in field and greenhouse tests for resistance to the Columbia root-knot nematode, Meloidogyne chitwoodi, as potential resistant crops in potato rotations. A nematode reproductive factor (Rf) was calculated for each cultivar. Reproductive factor values also were compared on a relative basis as percentages of the Rf on a susceptible field corn standard, Pioneer 3578. Popcorn cultivars W206 and Robust 33-77 consistently supported low population densities of M. chitwoodi in repeated tests. However, WOC 9508 had the greatest resistance in any of the field tests, with an Rf value of 0.04. Cultivars with a mean field and greenhouse Rf value less than 50% of the value for Pioneer 3578 were WOC 9508 (8%), WOC 9554 (13%), W206 (15%), WOX 9512 (23%), Robust 33-77 (30%), Robust 20-70 (38%), WOC 9510 (41%), and WOC 9504 (42%). If these cultivars were used in rotation, M. chitwoodi population densities at the end of the popcorn season would be between 58% and 92% less than if Pioneer 3578 were grown. In greenhouse tests, WOX 9511, WOX 9528, WOC 9556, and WOX 9531 also had low Rf values (7-46% that of Pioneer 3578), but field testing of these cultivars is needed.

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 degrees 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).

Characterization of Resistance in a Somatic Hybrid of Solanum bulbocastanum and S. tuberosum, to Meloidogyne chitwoodi.

A somatic hybrid, CBP-233, between resistant Solanum bulbocastanum (SB-22) and susceptible S. tuberosum (R4) was tested for resistance to Meloidogyne chitwoodi race 1. One week after inoculation, only 0.04-0.4% of the initial inoculum (Pi, 5,000 eggs) as second stage-juveniles infected SB-22 and CBP-233 root systems, compared to 2% in R4. After 8 weeks, the number of M. chitwoodi in SB-22 and CBP-233 roots remained lower (0.3-1.5% of Pi) compared to R4, which increased from 2% to ca. 27%. Development of M. chitwoodi was delayed on SB-22 and CBP-233 by at least 2 weeks, and only half of the infective nematodes established feeding sites and matured in resistant clones compared to 99% in susceptible R4. Necrotic tissue surrounded nematodes that failed to develop in SB-22 and CBP-233. The reproductive factor (ratio of final number of eggs recovered from roots to Pi) was <0.01 for both SB-22 and CBP-233 and 46.8 for R4. Delaying inoculation of CBP-233 from 1 to 3 months after planting did not increase the chance or rate of tuber infection. Only a few M. chitwoodi developed to maturity on CBP-233 tubers and deposited a small number of eggs. SB-22 rarely produced tubers in these experiments, and like CBP-233 were resistant to M. chitwoodi. It appeared that the mechanisms of resistance to M. chitwoodi in roots and tubers of CBP-233 are similar.

Influence of Temperature and Host Plant on the Interaction Between Pratylenchus neglectus and Meloidogyne chitwoodi.

The interaction between Pratylenchus neglectus (Pn) and Meloidogyne chitwoodi (Mc) was investigated at soil temperatures of 15, 20, and 25 C on barley and potato. Maximum numbers of Pn and Mc penetrated barley roots at 20 C, whereas a minimum number penetrated at 15 C. Pratylenchus neglectus restricted root penetration by Mc over time and vice-versa. Population densities of each species increased with increasing temperature. Concomitant inoculation of the two species resulted in lower numbers of Pn at 15 and 25 C in both barley and potato, whereas the numbers of Mc were lower at 15 C in barley and at 25 C in potato. Root weights of potato and barley at 15 and 20 C, respectively, were lowered by the presence of both nematodes singly or concomitantly. At 25 C, barley plants inoculated with Mc alone had lower shoot weight than uninoculated controls, but the damage was restricted when Pn also was present. The two species interact competitively, and the outcome varies with soil temperature and host plant. Pn has the potential to suppress Mc population levels and reduce the damage it causes to potato and barley.

Competition between the Plant-parasitic Nematodes Pratylenchus neglectus and Meloidogyne chitwoodi.

In experiments on competition between Pratylenchus neglectus and Meloidogyne chitwoodi in barley, the species that parasitized the roots first inhibited penetration by the latter species. Prior presence of P. neglectus impeded the development of M. chitwoodi. Pratylenchus neglectus reduced egg production, final population levels, and reproductive index of M. chitwoodi. The reduction was linearly related to initial population densities of P. neglectus. Initial population densities of M. chitwoodi had no effect on final population levels of P. neglectus. Carbon assimilation by barley plants was reduced when either nematode species was present alone, but not when both were present together. Both nematode species assimilated lower amounts of carbon when present together than when present alone. A split-root experiment demonstrated that translocatable chemicals were not involved in the competition between the two species.

Resistance of Diploid Triticeae Species and Accessions to the Columbia Root-knot Nematode, Meloidogyne chitwoodi.

The Columbia root-knot nematode, Meloidogyne chitwoodi race 2, is associated with several plant species, including members of the tribe Triticeae. We evaluated 15 diploid species for M. chitwoodi gall and reproductive indices from the following genera: Agropyron, Pseudoroegneria, Hordeum, Psathyrostachys, and Thinopyrum. Species from the genus Thinopyrum (Thinopyrum bessarabicum; J genome) and Psathyrostachys (Psathyrostachys fragilis, P. juncea, P. stoloniformis; N genome) expressed more resistance to M. chitwoodi than species within the genera Agropyron (Agropyron cristatum and A. mongolicum; P genome), Pseudoroegneria (Pseudoroegneria spicata, P. stipifolia, A. aegilopoicles, P. libanotica; S genome), and Hordeum (Hordeum bogdanii, H. brevisubulatum, H. californicum, and H. chilensis; H genome), although there was variation among individuals within P. spicata, P. juncea, and P. fragilis. The variation among genera and within species indicates that it would be possible to select Triticeae grasses for resistance to M. chitwoodi in order to identify and introgress genes for resistance into cultivated cereals.

Suppression of Meloidogyne chitwoodi with Sudangrass Cultivars as Green Manure.

Meloidogyne chitwoodi race 1 reproduced on Piper sudangrass (Sorghum bicolor (L.) Moench), 332 (sudangrass hybrid), and P855F and P877F (sorghum-sudangrass hybrids), but failed to reproduce efficiently on Trudan 8, Trudex 9 (sudangrass hybrids), and Sordan 79, SS-222, and Bravo II (sorghum-sudangrass hybrids). Meloidogyne chitwoodi race 2 behaved similarly and reproduced more efficiently on Piper, P855F, and P877F than on Trudan 8, Trudex 9, or Sordan 79. The mean reproductive factor for M. chitwoodi races on the poorer hosts ranged from <0.1 to 0.9 under greenhouse and field conditions. Meloidogyne hapla failed to reproduce on any of the cultivars tested. In the laboratory, leaves of each cultivar chopped and incorporated as green manure reduced the M. chitwoodi population in infested soil more than unamended or wheat green manure treatments. Trudan 8, although limited to the zone of incorporation, protected this zone from colonization of upward migrating second stage juveniles (J2) for up to 6 weeks. Leaves of Trudan 8 but not roots were effective against M. chitwoodi, and J2 appeared to be more sensitive than egg masses. Trudan 8 and Sordan 79 as green manure reduced M. chitwoodi in bucket microplots under field conditions.