Nematicidal Effects of Silver Nanoparticles on Root-knot Nematode in Bermudagrass.

Certain nematodes are common soilborne organisms found in turfgrass in the United States that cause significant economic damage to golf course turf. One of the most prevalent plant-parasitic nematodes infesting turfgrass are root-knot nematodes (Meloidogyne spp.). Chemical treatment options for root-knot nematodes in turfgrass are limited, and there is a need for new nematicidal active ingredients to address this problem. In this study, we evaluated the use of silver nanoparticles (AgNP) as a potential nematicide in laboratory and field experiments. AgNP was synthesized by a redox reaction of silver nitrate with sodium borohydride using 0.2% starch as a stabilizer. When J2 of M. incognita were exposed to AgNP in water at 30 to 150 µg/ml, >99% nematodes became inactive in 6 hr. When turfgrass and soil composite samples infested with M. graminis were treated with 150 µg/ml AgNP, J2 were reduced in the soil samples by 92% and 82% after 4- and 2-d exposures, respectively, in the treated compared to the nontreated soil samples. Field trials evaluating AgNP were conducted on a bermudagrass (Cynodon dactylon × C. transvaalensis) putting green infested with M. graminis. Biweekly application of 90.4 mg/m(2) of AgNP improved turfgrass quality in one year and reduced gall formation in the roots in two years without phytotoxicity. The AgNP application did not significantly reduce the number of M. graminis J2 in plots during the growing season. The laboratory assays attested to the nematicidal effect of AgNP, and the field evaluation demonstrated its benefits for mitigating damage caused by root-knot nematode in bermudagrass.

Expression of Phenylalanine Ammonia Lyase Genes in Maize Lines Differing in Susceptibility to Meloidogyne incognita.

Maize is a well-known host for Meloidogyne incognita, and there is substantial variation in host status among maize genotypes. In previous work it was observed that nematode reproduction increased in the moderately susceptible maize inbred line B73 when the ZmLOX3 gene from oxylipid metabolism was knocked out. Additionally, in this mutant line, use of a nonspecific primer for phenyl alanine ammonialyase (PAL) genes indicated that expression of these genes was reduced in the mutant maize plants whereas expression of several other defense related genes was increased. In this study, we used more specific gene primers to examine the expression of six PAL genes in three maize genotypes that were good, moderate, and poor hosts for M. incognita, respectively. Of the six PAL genes interrogated, two (ZmPAL3 and ZmPAL6) were not expressed in either M. incognita-infected or noninfected roots. Three genes (ZmPAL1, ZmPAL2, and ZmPAL5) were strongly expressed in all three maize lines, in both nematode-infected and noninfected roots, between 2 and 16 d after inoculation (DAI). In contrast, ZmPAL4 was most strongly expressed in the most-resistant maize line W438, was not detected in the most-susceptible maize line CML, and was detected only at 8 DAI in the maize line B73 that supported intermediate levels of reproduction by M. incognita. These observations are consistent with at least one PAL gene playing a role in modulating host status of maize toward M. incognita and suggest a need for additional research to further elucidate this association.

Inheritance of Resistance to Meloidoygne incognita in Primitive Cotton Accessions from Mexico.

Few sources of resistance to root-knot nematodes (Meloidogyne incognita) in upland cotton (Gossypium hirsutum) have been utilized to develop resistant cultivars, making this resistance vulnerable to virulence in the pathogen population. The objectives of this study were to determine the inheritance of resistance in five primitive accessions of G. hirsutum (TX1174, TX1440, TX2076, TX2079, and TX2107) and to determine allelic relations with the genes for resistance in the genotypes Clevewilt-6 (CW) and Wild Mexico Jack Jones (WMJJ). A half-diallel experimental design was used to create 28 populations from crosses among these seven sources of resistance and the susceptible cultivar DeltaPine 90 (DP90). Resistance to M. incognita was measured as eggs per g roots in the parents, F(1) and F(2) generations of each cross. The resistance in CW and WMJJ was inherited as recessive traits, as reported previously for CW, whereas the resistance in the TX accessions was inherited as a dominant trait. Chi square analysis of segregation of resistance in the F(2) was used to estimate the numbers of genes that conditioned resistance. Resistance in CW and WMJJ appeared to be a multigenic trait whereas the resistance in the TX accessions best fit either a one or two gene model. The TX accessions were screened with nine SSR markers linked to resistance loci in other cotton genotypes. The TX accessions lacked the allele amplified by SSR marker CR316 and linked to resistance in CW and other resistant genotypes derived from this source. Four of five TX genotypes lacked the amplification products from the marker BNL1231 that is also associated with the resistant allele on Chromosome 11 in WMJJ, CW, NemX, M120 RNR and Auburn 634 RNR. However, all five TX genotypes produced the same amplification products from three SSR markers linked to the resistant allele on Chromosome 14 in M120 RNR and M240 RNR. The TX accessions have unique resistance genes that are likely to be useful in efforts to develop resistant cotton cultivars with increased durability.

Reproduction of Meloidogyne marylandi and M. incognita on several Poaceae.

The susceptibility of 22 plant species to Meloidogyne marylandi and M. incognita was examined in three greenhouse experiments. Inoculum of M. marylandi was eggs from cultures maintained on Zoysia matrella "Cavalier" or Cynodon dactylon x C. trasvaalensis "Tifdwarf". Inoculum of M. incognita was eggs from cultures maintained on Solanum lycopersicum 'Rutgers'. In each host test the inoculum density was 2,000 nematode eggs/pot. None of the three dicot species tested (Gossypium hirsutum, Arachis hypogaea, and S. lycopersicum) were hosts for M. marylandi but, as expected, M. incognita had high levels of reproduction on G. hirsutum and S. lycopersicum. Meloidogyne marylandi reproduced on all of the 19 grass species (Poaceae) tested but reproduction varied greatly (P = 0.05) among these hosts. The following grasses were identified for the first time as hosts for M. marylandi: Buchloe dactyloides (buffalograss), Echinochloa colona (jungle rice), Eragostis curvula (weeping lovegrass), Paspalum dilatatum (dallisgrass), P. notatum (bahiagrass), Sorghastrum, nutans (indiangrass), Tripsacum dactyloides (eastern gamagrass), and Zoysia matrella (zoysiagrass). No reproduction of M. incognita was observed on B. dactyloides, Cyndon dactylon (common bermudagrass), E. curvula, P. vaginatum (seashore paspalum), S. nutans, T. dactyloides, Z. matrella or Z. japonica. Reproduction of M. incognita was less than reproduction of M. marylandi on the other grass species, except for the Zea mays inbred line B73 on which M. incognita had greater reproduction than did M. marylandi (P = 0.05) and Stenotaphrum secundatum (St. Augustinegrass) on which M. incognita and M. marylandi had similar levels of reproduction.

Cotton root protection from plant-parasitic nematodes by abamectin-treated seed.

Abamectin is nematicidal to Meloidogyne incognita and Rotylenchulus reniformis, but the duration and length of cotton taproot protection from nematode infection by abamectin-treated seed is unknown. Based on the position of initial root-gall formation along the developing taproot from 21 to 35 d after planting, infection by M. incognita was reduced by abamectin seed treatment. Penetration of developing taproots by both nematode species was suppressed at taproot length of 5 cm by abamectin-treated seed, but root penetration increased rapidly with taproot development. Based on an assay of nematode mobility to measure abamectin toxicity, the mortality of M. incognita associated with a 2-d-old emerging cotton radicle was lower than mortality associated with the seed coat, indicating that more abamectin was on the seed coat than on the radicle. Thus, the limited protection of early stage root development suggested that only a small portion of abamectin applied to the seed was transferred to the developing root system.

The future of nematode management in cotton.

The importance of plant-parasitic nematodes as yield-limiting pathogens of cotton has received increased recognition and attention in the United States in the recent past. This paper summarizes the remarks made during a symposium of the same title that was held in July 2007 at the joint meeting of the Society of Nematologists and the American Phytopathological Society in San Diego, California. Although several cultural practices, including crop rotation, can be effective in suppressing the populations of the important nematode pathogens of cotton, the economic realities of cotton production limit their use. The use of nematicides is also limited by issues of efficacy and economics. There is a need for development of chemistries that will address these limitations. Also needed are systems that would enable precise nematicide application in terms of rate and placement only in areas where nematode population densities warrant application. Substantial progress is being made in the identification, characterization and mapping of loci for resistance to Meloidogyne incognita and Rotylenchulus reniformis. These data will lead to efficient marker-assisted selection systems that will likely result in development and release of nematode-resistant cotton cultivars with superior yield potential and high fiber quality.

Modeling manure-borne bromide and fecal coliform transport with runoff and infiltration at a hillslope.

Hillslope vegetated buffers are recommended to prevent water pollution from agricultural runoff. However, models to predict the efficacy of different grass buffer designs are lacking. The objective of this work was to develop and test a mechanistic model of coupled surface and subsurface flow and transport of bacteria and a conservative tracer on hillslopes. The testing should indicate what level of complexity and observation density might be needed to capture essential processes in the model. We combined the three-dimensional FEMWATER model of saturated-unsaturated subsurface flow with the Saint-Venant model for runoff. The model was tested with data on rainfall-induced fecal coliforms (FC) and bromide (Br) transport from manure applied at vegetated and bare 6-m long plots. The calibration of water retention parameters was unnecessary, and the same manure release parameters could be used both for simulations of Br and FC. Surface straining rates were similar for Br and bacteria. Simulations of Br and FC concentrations were least successful for the funnels closest to the source. This could be related to the finger-like flow of the manure from the strip along the bare slopes, to the transport of Br and FC with manure colloids that became strained at the grass slope, and to the presence of micro-ponds at the grassed slope. The two-dimensional model abstraction of the actual 3D transport worked well for flux-averaged concentrations. The model developed in this work is suitable to simulate surface and subsurface transport of agricultural contaminants on hillslopes and to evaluate efficiency of grass strip buffers, especially when lateral subsurface flow is important.

Suppression of Rotylenchulus reniformis 122-cm Deep Endorses Resistance Introgression in Gossypium.

Nine sources of resistance to Rotylenchulus reniformis in Gossypium (cotton) were tested by measuring population density (Pf) and root-length density 0 to 122 cm deep. A Pf in the plow layer less than the autumn sample treatment threshold used by consultants was considered the minimum criterion for acceptable resistance, regardless of population density at planting (Pi). Other criteria were ample roots and a Pf lower than on the susceptible control, as in pot studies. In a Texas field in 2001 and 2002, no resistant accessions had Pf less than the control but all did in microplots into which nematodes from Louisiana were introduced. An environmental chamber experiment ruled out nematode genetic variance and implicated unknown soil factors. Pf in field experiments in Louisiana, Mississippi, and Alabama were below threshold for zero, six and four of the accessions and above threshold in the control. Gossypium arboreum A2-87 and G. barbadense GB-713 were the most resistant accessions. Results indicate that cultivars developed from these sources will suppress R. reniformis populations but less than in pots in a single season.

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 LD(50) 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, LD(50) values of 1.56 mug/ml and 32.9 mug/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 mug/ml for M. incognita and 32.9 to 8.2 mug/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.

Improving the utility of nematode resistance in groundnut.

Host resistance to pests and pathogens, to be useful, must be introgressed into cultivars that possess the high yield potential and other important agronomic quality traits desired by the growers. The root-knot nematodes M. arenaria and M. javanica are only a subset of the several important pests and pathogens attacking groundnut. The resistance that was transferred from wild Arachis spp. to cultivated groundnut will provide nearly complete control of these important pathogens; however, growers have been reluctant to use this resistance because the resistant cultivars lack other desired traits. The introgression of nematode resistance into genotypes that have multiple disease resistance, higher yield potential, and the important agronomic trait of the high O/L ratio will provide growers with a cultivar that satisfies most of their needs. Our current efforts are aimed at providing growers with high quality cultivars with high levels of resistance to multiple diseases. Development of germplasm with these multiple traits will ensure that potential benefits of host resistance are realized.

Fate of dietary perchlorate in lactating dairy cows: Relevance to animal health and levels in the milk supply.

Perchlorate is a goitrogenic anion that competitively inhibits the sodium iodide transporter and has been detected in forages and in commercial milk throughout the U.S. The fate of perchlorate and its effect on animal health were studied in lactating cows, ruminally infused with perchlorate for 5 weeks. Milk perchlorate levels were highly correlated with perchlorate intake, but milk iodine was unaffected, and there were no demonstrable health effects. We provide evidence that up to 80% of dietary perchlorate was metabolized, most likely in the rumen, which would provide cattle with a degree of refractoriness to perchlorate. Data presented are important for assessing the environmental impact on perchlorate concentrations in milk and potential for relevance to human health.

A Recessive Gene for Resistance to Meloidogyne arenaria in Interspecific Arachis spp. Hybrids.

A single dominant gene for resistance to Meloidogyne arenaria was identified previously in two peanut cultivars, Arachis hypogaea 'COAN' and 'NemaTAM'. The interspecific Arachis hybrid TxAG-6 was the source of this resistance and the donor parent in a backcross breeding program to introgress resistance into cultivated peanut. To determine if other resistance genes were present in TxAG-6 and derived breeding populations from the third backcross generation (BC), F individuals were evaluated for the resistance phenotype. The ratio of the resistant and susceptible individuals for all F populations fit the expected ratio for resistance being governed by one dominant gene and one recessive gene. Evaluation of the F generation from four susceptible F individuals (two from TxAG-6 x A. hypogaea and two from the BC population) confirmed that a recessive gene for resistance to M. arenaria was present in each of the tested populations. The identification of a second gene for resistance in the A. hypogaea germplasm may improve the durability of the resistance phenotype.

Laser Capture Microdissection and Real-Time PCR for Measuring mRNA in Giant Cells Induced by Meloidogyne javanica.

The techniques of laser capture microdissection and quantitative RT-PCR were investigated as methods for measuring mRNA in giant cells induced by Meloidogyne javanica. Laser capture microdissection allowed precise sampling of giant cells at 1 to 3 weeks after inoculation. The expression of three genes (a water channel protein gene Rb7, a plasma membrane H(+)-ATPase (LHA4), and a hexose kinase (HXK1) was measured based on mRNA extracted from tissue samples and quantitated using reversetranscription real-time PCR. These genes were chosen arbitrarily to represent different aspects of primary metabolism. The amount of HXK1 mRNA in giant cells was not different from that in root meristem or cortical cells when compared on the basis of number of molecules per unit tissue volume, and was similar at all sample times. Amount of mRNA for LHA4 and Rb7 was much greater in giant cells than in cortical cells, but only Rb7 was also greater in giant cells than in root meristem cells. Numbers of mRNA molecules of LHA4 increased linearly in giant cells from 1 to 3 weeks after inoculation, whereas the amount of Rb7 mRNA was similar at 1 and 2 weeks after inoculation but increased at 3 weeks after inoculation. The amount of mRNA for these two genes was similar at all sample times in cortical and root-tip cells. Apparent up regulation of some genes in giant cells may be due primarily to the increased number of copies of the gene in giant cells, whereas for other genes up regulation may also involve increased transcription of the increased number of copies of the gene.

Meloidogyne haplanaria n. sp. (Nematoda: Meloidogynidae), a Root-knot Nematode Parasitizing Peanut in Texas.

Meloidogyne haplanaria n. sp. is described and illustrated from specimens parasitizing peanut in Texas. The perineal pattern of the female is rounded to oval with a dorsal arch that is high and rounded except for striae near the vulva, which are low with rounded shoulders. The striae are distinctly forked in the lateral field, and punctations often occur as a small group near the tail tip and singly within the whole perineal pattern. The female stylet is 13-16 microm long and has broad, distinctly set-off knobs. The excretory pore opens 40-118 microm from the head, approximately halfway between the anterior end and the metacorpus. Males are 1.2-2.4 microm in length and have a high, wide head cap that slopes posteriorly. The labial disc and medial lips are partially fused to form an elongated lip structure. In some specimens the labial disk is distinctly separated from the lips by a groove. The stylet is 17-22 microm long and has wide knobs that are rounded and distinctly set off from the shaft. Mean second-stage juvenile length is 419 microm. The head region is not annulated, and the large labial disc and crescent-shaped medial lips are fused to form a dumbbell-shaped head cap. The stylet is 9-12 microm long and has rounded, posteriorly sloping knobs. The slender tail, 58-74 microm long, has a distinct, inflated rectum and a slightly rounded tip. The hyaline tail terminus is 11-16 microm long. The isozyme phenotypes for esterase and malic dehydrogenase do not correspond to any other recognized Meloidogyne species. Tomato and peanut are good hosts; corn and wheat are very poor hosts; and cotton, tobacco, pepper, and watermelon are nonhosts.

Mechanism of Resistance to Meloidogyne arenaria in the Peanut Cultivar COAN.

Resistance to Meloidogyne arenaria in the peanut cultivar COAN is inherited as a single, dominant gene. The mechanism of resistance to M. arenaria in COAN was evaluated in three experiments. In the first experiment the number of second-stage juveniles (J2) of M. arenaria penetrating roots of the susceptible cultivar Florunner was higher than the number of J2 penetrating roots of the resistant peanut cultivar COAN (P < 0.05). In a second experiment it was determined that the root size and number of potential infection courts (root tips) were similar for the two peanut cultivars. The number of nematodes emigrating from roots of COAN after penetration was greater than emigrated from roots of Florunner (P < 0.05). Necrotic host tissue was rarely observed in roots of COAN infected with M. arenaria, suggesting that resistance to M. arenaria does not involve a necrotic, hypersensitive response. Most of the J2 observed in roots of COAN were restricted to the cortical tissue, with only 1 of 90 J2 observed being associated with the vascular cylinder, whereas in Florunner >70% of the J2 were associated with vascular tissues. Resistance in COAN may be due to constitutive factors in the roots.

Transmission genetics of chromatin from a synthetic amphidiploid to cultivated peanut (Arachis hypogaea L.). broadening the gene pool of a monophyletic polyploid species.

Polyploidy creates severe genetic bottlenecks, contributing to the genetic vulnerability of leading crops. Cultivated peanut is thought to be of monophyletic origin, harboring relatively little genetic diversity. To introduce variability from diploid wild species into tetraploid cultivated Arachis hypogaea, a synthetic amphidiploid [[A. batizocoi K9484 x (A. cardenasii GKP10017 x A. diogoi GKP10602)](4x)] was used as donor parent to generate a backcross population of 78 progeny. Three hundred seventy RFLP loci were mapped onto 23 linkage groups, spanning 2210 cM. Chromatin derived from the two A-genome diploid ancestors (A. cardenasii and A. diogoi) comprised mosaic chromosomes, reflecting crossing over in the diploid A-genome interspecific F(1) hybrid. Recombination between chromosomes in the tetraploid progeny was similar to chromosome pairing reported for A. hypogaea, with recombination generally between chromosomes of the same subgenomic affinity. Segregation distortion was observed for 25% of the markers, distributed over 20 linkage groups. Unexpectedly, 68% of the markers deviating from expected segregation showed an excess of the synthetic parent allele. Genetic consequences, relationship to species origins, and significance for comparative genetics are discussed.

Root Galling and Reproduction of Meloidogyne incognita Isolates from Texas on Resistant Cotton Genotypes.

Several cotton genotypes with resistance to Meloidogyne incognita have been released in recent years. To estimate the durability of this resistance, galling severity on these resistant genotypes by M. incognita was measured. Nematode isolates (115 total) were collected from cotton fields in 14 Texas counties in August and September 1996 and 1997. Four additional isolates from Maryland, Mississippi, and North Carolina were also tested. The isolates were evaluated in 12 greenhouse experiments for their ability to gall roots of the resistant cotton genotypes M315, Acala NemX, and Stoneville LA887 and the susceptible cultivar Deltapine 90. Numbers of galls on each genotype by each isolate were counted 60 days after inoculation with 10,000 eggs/plant. M315 consistently had the fewest galls for each nematode isolate, whereas Deltapine 90 had the greatest number of galls. Numbers of galls on NemX and LA887 were usually intermediate and more variable. For each separate experiment, analysis of variance indicated that the effects of nematode isolates, cotton genotypes, and isolate-genotype interaction were significant (P < 0.05). In two of the experiments, nematode reproduction was also measured and galling was positively correlated (r = 0.68 and 0.86) with egg production by M. incognita. Nematode isolates from one field exhibited higher root galling and reproduction (P < 0.05) on resistant genotypes than other isolates, suggesting a need for gene deployment systems that will enhance the durability of resistance.

Genetics and Mechanism of Resistance to Meloidogyne arenaria in Peanut Germplasm.

Segregation of resistance to Meloidogyne arenaria in six BCF peanut breeding populations was examined in greenhouse tests. Chi-square analysis indicated that segregation of resistance was consistent with resistance being conditioned by a single gene in three breeding populations (TP259-3, TP262-3, and TP271-2), whereas two resistance genes may be present in the breeding populations TP259-2, TP263-2, and TP268-3. Nematode development in clonally propagated lines of resistant individuals of TP262-3 and TP263-2 was compared to that of the susceptible cultivar Florunner. Juvenile nematodes readily penetrated roots of all peanut genotypes, but rate of development was slower (P = 0.05) in the resistant genotypes than in Florunner. Host cell necrosis indicative of a hypersensitive response was not consistently observed in resistant genotypes of either population. Three RFLP loci linked to resistance at distances of 4.2 to 11.0 centiMorgans were identified. Resistant and susceptible alleles for RFLP loci R2430E and R2545E were quite distinct and are useful for identifying individuals homozygous for resistance in segregating populations.

Resistance of Interspecific Arachis Breeding Lines to Meloidogyne javanica and an Undescribed Meloidogyne Species.

Resistance to a peanut-parasitic population of Meloidogyne javanica and an undescribed Meloidogyne sp. in peanut breeding lines selected for resistance to Meloidogyne javanica was examined in greenhouse tests. The interspecific hybrid TxAG-7 was resistant to reproduction of Meloidogyne javanica, M. javanica, and Meloidogyne sp. An Meloidogyne javanica-resistant selection from the second backcross (BC) of TxAG-7 to the susceptible cultivar Florunner also was resistant to M. javanica but appeared to be segregating for resistance to the Meloidogyne sp. When reproduction of M. javanica and Meloidogyne javanica were compared on five BC4F3 peanut breeding lines, each derived from Meloidogyne javanica-susceptible BC4F2 individuals, all five lines segregated for resistance to M. javanica, whereas four of the lines appeared to be susceptible to Meloidogyne javanica. These data indicate that several peanut lines selected for resistance to Meloidogyne javanica also contain genes for resistance to populations of M. javanica and the undescribed Meloidogyne sp. that are parasitic on peanut. Further, differences in segregation patterns suggest that resistance to each Meloidogyne sp. is conditioned by different genes.

Distribution of Isolates of Sclerotium rolfsii Tolerant to Pentachloronitrobenzene in Texas Peanut Fields.

The tolerance to pentachloronitrobenzene (PCNB) of an isolate of Sclerotium rolfsii collected in 1985 was quantified, and a survey of tolerance to PCNB in 377 other isolates of the fungus collected from Texas peanut fields from 1990 through 1994 was conducted. The effective dose (ED)50 of the previously collected PCNB-tolerant isolate was 11.07 µg PCNB/ml and was more than 5-fold greater than the ED50 of PCNB-sensitive isolates. The distribution of tolerance to PCNB among all isolates was slightly skewed, with 18 of the 377 isolates identified as having greater (P ≤ 0.05) tolerance to PCNB than the standard sensitive isolate. No isolate of S. rolfsii collected during the period of 1990 to 1994 had as high an ED50 value as did the 1985 isolate, even among those isolates collected from the same field from which the 1985 isolate was collected. ED50 values of two PCNB-sensitive and five PCNB-tolerant isolates were unchanged after 15 generations on potato dextrose agar amended with 10 µg PCNB/ml or on unamended media. The PCNB-tolerant isolate collected in 1985 was less aggressive than other isolates in greenhouse and microplot tests, but no correlation was observed between ED50 values and disease incidence in these tests for other PCNB-sensitive and tolerant isolates. These data suggest that even though high levels of tolerance to PCNB can be confirmed in some isolates of S. rolfsii, this phenomenon is likely to remain a rare event.