The great majority of engineered crops in agriculture and in our food supply are Roundup Ready. Roundup Ready crops are engineered to be sprayed with the herbicide Roundup and this technology has become so successful that Roundup has become a major pollutant. The active ingredient of the herbicide Roundup is glyphosate, a molecule created in 1960 to clean industrial pipes and boilers of mineral scales (i.e., mineral deposits). Glyphosate was initially patented as a powerful and broad-spectrum descaling agent. It binds indiscriminately and does a great job at dissolving and preventing minerals from being bioavailable. We have plenty of evidence that this chemical property also makes this molecule a biocide. Glyphosate kills bacteria at 1 ppm and is an endocrine disruptor in animals at 0.5 ppm. Glyphosate depletes minerals in soil, in plants and in animals that eat the plants.

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.

The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida.

Population densities of Pratylenchus penetrans and the biomass of fine roots of raspberry at depths of 0-5, 5-10, 10-20, and 20-30 cm were determined every 2 weeks for 2 years. The vertical distribution of P. penetrans varied from season to season, but the seasonal changes were not similar for the 2 years. In most seasons, the greatest population density was in the 5 to 10-cm-depth interval. Population densities of P. penetrans were not consistently correlated with the vertical distribution of raspberry roots in any season.

The root-lesion nematode Pratylenchus penetrans is a severe pathogen of raspberry (Rubus idaeus L.) and other small fruit crops. Temporal changes in the size and distribution between root and soil habitats of a population of dhe nematode under raspberry were monitored. Soil and root samples were collected at four depths from five plots in an 8-year-old raspberry planting every 2 weeks for 2 years. Trends of nematode densities were not identical in the 2 years. There were no sustained changes in the fall and winter of the first year, a reduction in nematode population in the first spring followed by increases in early summer and early fail followed by a decrease in late fall. There were no sustained changes in the second year except fur a single reduction in late spring and summer. The proportion of the nematode population in roots ranged from a maximum of 0.69 to a minimum of 0.07, but that proportion did not follow a seasonal pattern. Nematode densities were not correlated with root abundance. Because of the lack of regular seasonal population trends, it is suggested thai parameters other than temperature and root availability may have controlled reproduction of the nematodes, and distribution between roots and rhizosphere soil of perennial crops.

Four steinernematid strains, Steinernema carpocapsae All strain, S. carpocapsae Umea strain, S. feltiae L1c strain, and S. feltiae strain 27, were evaluated in laboratory and field tests as potential agents for the control of the black army cutworm, Actebia fennica (Tauscher). Although each of the six instars was susceptible to attack, incidences of infection were highest and generally over 60% for the third, fourth, and fifth instars when exposed to 100 infective juvenile nematodes in petri dishes. Fifth-instar larvae exposed for 1 week to soil containing 4000 infective juveniles in a 1:1:1 mixture of the Lie, All, and Umea strains were most frequently infected with the Lie strain when reared at 10 and 12 degrees C and with the All strain when reared at 14 and 16 degrees C. One hundred thousand infective juveniles of the Lie, All, and Umea strains and strain 27, alone and in various combinations, and applied by either a spray or soil-plug inoculation, killed black army cutworm larvae caged in the field for 1 week with treated black spruce seedlings and provided protection of current and 1-year-old foliage. Treatments that included strain 27 provided the best protection when daily mean soil temperatures were 7-11 degrees C in comparison to treatments that included the Umea or All strains, whereas treatments that included the All strain as a component appeared to be more effective than treatments containing the Lie and Umea strains when daily mean soil temperatures were 16-17 degrees C.

Cover crops used in red raspberry plantings (Rubus idaeus L.) are often good hosts of the root-lesion nematode (Pratylenchus penetrans Filipjev & Sch. Stekoven), a major soilborne pathogen of raspberry. The effects of two susceptible cover crops, white clover (Trifolium repens L.) and barley (Hordeum vulgare L.), planted in between rows, on nematode density and growth of raspberry plants were compared to those of three cover crops resistant to the nematode: redtop (Agrostis alba L.), creeping red fescue (Festuca rubra L.), and 'Saia' oat (Avena sativa L.), Nematode multiplication in raspberry roots and in cover crop roots was assessed over 4 years, Growth and vigor of plants were estimated at the end of the experiment by counting primocanes and determining height and biomass. Nematode multiplication was suppressed in roots of 'Saia' oat, fescue, and redtop compared to barley or white clover. Nematode density in roots and rhizosphere soil of raspberry was not affected by the choice of cover crops. Nematode suppression in the three resistant cover crops did not translate into increased vigor of raspberry plants.

A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the K-i values), at least with the disulfide bond-lacking cystatins. Complexes with K-i values greater than or equal to 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower K-i values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest - the two-spotted spider mite (Tetranychus urticate Koch). the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases. without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans.

The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla. M. incognita and M. javanica , were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second‐stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans ‐epoxysuccinyl‐L‐leucylamido‐(4‐guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class‐specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla , and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition by OCI was partly prevented during electrophoresis. This species‐related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne .

The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Otiorynchus sulcatus, were assessed using complementary inhibition assays, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 microM) caused partial and stable inhibition of total proteolytic (azocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large excess (100 microM). As demonstrated by complementary inhibition assays, this two-step inhibition of the insect proteases by HSA was due to the differential inactivation of two distinct cysteine proteinase populations in either insect extracts, the rapidly (strongly) inhibited population corresponding to the OCI-sensitive fraction. After removing the cystatin-sensitive proteinases from CPB and BVW midgut extracts using OCI- (or HSA-) affinity chromatography, the effects of the insect "non-target" proteases on the structural integrity of the two cystatins were assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermediates, suggesting the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of HSA to proteolytic cleavage may partially explain its low efficiency to inactivate the insect OCI-insensitive cysteine proteinases when not used in large excess. It could also have major implications when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests.

A new Ditylenchus species from South Africa that parasitizes peanut is described based on characteristics of morphology and restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA). The new species, Ditylenchus africanus sp. n., is different from the two most similar species, D. destructor and D. myceliophagus by the following combination of characters : RFLP's generated by seven restriction enzymes on the internal transcribed spacer of rDNA, a medium long, relatively weak stylet (compared with the stylet of D. destructor), bursa length (as percentage of tail length) and spicule length.

The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a ∼10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearence of OCII resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control.

The inheritance of resistance to the root lesion nematode [Pratylenchus penetrans (Cobb) Filip. and Stek.] in red raspberry (Rubus idaeus L.) was studied in a four-member half diallel, involving two resistant genotypes and two susceptible genotypes. Estimates of general and specific combining abilities (GCA and SCA, respectively) were determined for nematode densities in roots alone and soil alone, nematode densities per plant, and plant root and foliage biomass. GCA were significant for nematodes in soil and for root and foliage biomass; SCA were significant for nematodes in the soil and for root biomass. Neither GCA nor SCA was significant for number of nematodes in the roots or per plant.

This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron.

Growth, dry root weight of seedlings and root score of apple seedlings cv. McIntosh were reduced when soils were inoculated with Pratylenchus penetrans, Penicillium janthinellum, Constantinella terrestris, Trichoderma sp., and 4 strains of Bacillus subtilis. Trichoderma sp., and B-1 and B-26 strains of B. subtilis alone reduced plant growth but the combination of Trichoderma sp. + B. subtilis (B-1) and Trichoderma sp. + B. subtilis (B-26) increased plant height. Plant height, root weight and root score were significantly reduced when P. penetrans plus B. subtilis or P. penetrans plus fungi plus bacteria were present in the soil. It is suggested that fungi, bacteria, nematodes alone or their combinations such as nematodes plus bacteria or nematodes plus fungi plus bacteria may contribute towards the occurrence of apple replant disease.

Xiphinema bricolensis n.sp. and Xiphinema pacificum n.sp. are described and illustrated from British Columbia vineyards. Xiphinema bricolensis differs from X. occiduum by the longer odontostyle, longer tail, smaller a value, and larger c′ value. It differs from X. thornei by the smaller c value, larger c′ value, longer tail, and greater anal body width. Xiphinema pacificum is closely related to X. californicum but differs by the longer tail, larger c′ value, longer odontophore, more posterior vulva, and position of the caudal papillae.