First report of, and additional information on, Meloidogyne konaensis (Nematoda: Meloidogyninae) parasitising various crops in Brazil

In a survey for Meloidogyne spp. in different crops from 11 regions in Ceará State, Brazil, using esterase isozyme electrophoresis as a specific identification method, four atypical populations were characterised from cabbage, papaya, noni and canapum plants, all of which showed an esterase profile different from those previously detected in Brazil. Morphological studies showed typical characteristics of Meloidogyne konaensis. Perineal patterns of females were variable, similar to M. arenaria and M. incognita, stylet length 14-20 μm. In females, the knobs gradually merged with the shaft and the dorsal pharyngeal gland orifice (DGO) ranged from 4 to 7 μm. Although males are not frequently found, the stylet morphology provides the most useful source of diagnostic character for the species, having 6-12 large projections protruding from the shaft. The esterase pattern K3 is unique and species-specific with three major bands Rm 1.0, 1.17, 1.27 and a secondary band Rm 1.10. Some confusion about the true identity of this species was clarified in this study, including differentiation from M. paranaensis. A species-specific SCAR marker developed for M. paranaensis was tested and no amplification products were observed. In Neighbour-Joining analyses of ITS and D2-D3 rRNA sequences, M. konaensis from Brazil appeared clearly separated from M. paranaensis. Pathological tests indicated that coffee is not a host of M. konaensis as previously reported in the original description of this species.

Isoenzymatic and cytological studies of some Asiatic species of the genus Salsola

The genetic and cytological variability of population of three <em>Salsola</em> species from Asia was investigated, using isozyme electrophoresis and haematoxylin staining. Eight enzyme systems, representing 14-17 loci, were examined: 6PGD, DIA, G6PD, GDH, GOT, MDH, PGM and PGI. Analysis of the chromosome number revealed that the three species have the same number of chromosomes: <em>2n=18</em>. Parameters describing genetic diversity indicate a very low level of genetic variation of the studied populations. The isozyme data support hypothesis that strong directional selection can result in lower level of genetic variation of arid plant populations.

First Report of the Root-Knot Nematode Meloidogyne artiellia in Belgium

In 2011, second-stage juveniles (J2) of an unknown root-knot nematode (Meloidogyne spp.) were detected during a routine survey for root-knot nematodes on arable land in Harveng, Belgium, after a crop of wheat. Most of the loamy soil samples (36 out of 42) contained J2 of the common root-knot nematode M. naasi Franklin, 1965 (1), while 15 of these also contained the unknown species, albeit in lower densities (22 J2/100 ml vs. 157 J2/100 ml soil). After detailed morphological observation of the unknown J2, they were until further notice identified as Meloidogyne artiellia Franklin, 1961 (2), the British root-knot nematode. To confirm the identification, a pure culture of M. artiellia was established by adding nematode suspensions to pots planted with kale (Brassica oleracea var. laciniata), a non-host for M. naasi (3). After 2 months, Meloidogyne spp. females, males, and J2 were isolated from galled kale roots. Morphological characteristics (n = 25) from the perineal pattern (rounded with fine striae, lateral area with coarse ridges, angular dorsal arch) and stylet knobs (small, ovoid, and backwardly sloping) for the females, the head shape (set off with distinct head cap) and stylet knobs (small, ovoid and backwardly sloping) for the males, the hemizonid position (anterior, adjacent to S to E pore), tail shape (conical), and short tail length (18 to 27 μm) for the J2, fit with previous observed populations of M. artiellia (3). Young egg-laying females were used for isozyme electrophoresis, and showed typical malate dehydrogenase (N1b) and esterase (M2-VF1) patterns (3). Additionally, DNA was extracted from single juveniles by incubating them in a lysis buffer (200 mM NaCl, 200 mM Tris-HCl (pH 8), 1% β-mercaptoethanol and 800 μg/ml Proteinase K) during 1.5 h at 65°C and 5 min at 99°C in a thermocycler. One microliter of crude DNA extract was used for PCR. ITS-rDNA sequencing (GenBank Accession Numbers JX393299 and JX393300) confirmed the identity, showing a 98 to 100% homology with other M. artiellia sequences (AY150368 and AF248478). To our knowledge, this is the first report of the root-knot nematode, M. artiellia, in Belgium. This nematode has been reported from the Mediterranean area, where it causes damage on chickpea and wheat (4), as well as from the U.K. Its finding in Harveng, close to the French border, suggests a more extensive geographical distribution. References: (1) M. T. Franklin. Nematologica 11:79, 1965. (2) M. T. Franklin et al. Suppl.:85, 1961. (3) G. Karssen. Pages 93-97 in: The Plant-Parasitic Nematode Genus Meloidogyne Göldi, 1892 (Tylenchida) in Europe, Brill Leiden, The Netherlands, 2002. (4) M. Di Vito and N. Greco. Revue Nématol. 11:223, 1988.

Latitudinal variation of genecological traits in native grasses of Patagonian rangelands

Geographical variation in genetically based traits helps to elucidate the effect of distinct ecological and evolutionary processes on widespread plants. Whereas abundant information exists on genetic patterns of woody species in western humid Andes, such information is scarce for the neighbouring dry Patagonian steppe. We examined genecological traits of two native forage species vulnerable to overgrazing (Bromus pictus and Poa ligularis) in dry Occidental Phytogeographical District. We compared within-population genetic diversity and among-population (n = 6) divergence by using isozyme electrophoresis. We also cultivated plants under common garden to compare genetically based morphology (plant height, number of tillers by plant and weight per tiller). Analysis showed that 8 and 13 loci were polymorphic of 9 and 19 resolved loci in at least one population for Bromus and Poa, respectively. In general, plant traits decreased from north to south in both species. Genetic and quantitative results (FST/QST index) showed evidence of local adaptation in populations of both species. Genetic divergence among populations was significant. We detected two different geographical groups divided at the same latitude (42–43°S) in both species, supporting the hypothesis of a past vicariance event. Sustainable management of these forage species to cope with land-use and climate change will be enriched by the inclusion of genecological knowledge.

A Study on the Toxicity of a Medicinal Plant, Artemisia Annua L. (Asteracea) Extracts to the Sunn Pest, Eurygaster Integriceps Puton (Hemiptera: Scutelleridae)

A Study on the Toxicity of a Medicinal Plant,Artemisia AnnuaL. (Asteracea) Extracts to the Sunn Pest,Eurygaster IntegricepsPuton (Hemiptera: Scutelleridae)A methanolic extract ofArtemisia annuawas obtained to evaluate its insecticidal activities against the sunn pest (Eurygaster intefriceps). Also, the responses of general esterase (EST), glutathione S-transferase (GST), alkaline phosphatase (ALP), acid phosphatase (ACP), acethylcholinesterase (AChE) to the plant extract were investigated. Topical application of plant extract on adults showed that the mortality was dose-dependent i.e. with increasing of plant extract concentrations more mortality achieved. Esterase and GST activities were increased in the first 24 h post-treatment. However, the enzymes activities were decreased after 24 h until 72 h. The activities of ALP, ACP and AChE in insect body decreased significantly and inhibition was higher along with increasing concentrations of plant extract. Isozyme electrophoresis profiles indicated that responses of isozymes (EST and GST) to plant extract were decreased after 48 h exposure to extract so that some enzymes bands disappeared. The results indicated that the highest concentration ofA. annuaextract was the most toxic among the four extracts. The decline of the detoxification ability in insects' tissues might be the main reason for the insecticidal activities.

Superoxide dismutase isoelectric focusing patterns as a tool to differentiate pathotypes of Globodera spp.

Isoelectric focusing was used to separate proteins from cyst extracts of potato cyst nematode (PCN) populations. In a first set of assays, cyst extracts from standard populations of Globodera rostochiensis pathotypes Ro1, Ro2, Ro3, Ro2/3, Ro4, and Ro5, and G. pallida pathotypes Pa2 and Pa3, were loaded on isoelectric focusing gels. Gels were stained for superoxide dismutase (SOD), esterase, and glucose-6-phosphate isomerase (GPI). Twelve bands of SOD activity were detected, six (B1-B6) migrating towards the basic zone and the other six (A1-A6) migrating towards the acidic zone, starting from the loading point. A cluster analysis was carried out based on a data matrix that reported the presence or absence of SOD bands on the isozyme electrophoresis patterns (IEPs). Globodera spp. were clearly distinguished and, within G. rostochiensis, Ro2 and Ro4 shared a high level of similarity, respectively, with Ro3 and Ro5; moreover, Ro1 could be clearly distinguished from Ro2/3 and Ro4/5. Globodera pallida Pa2 and Pa3 also shared a high level of similarity. In contrast, esterase and GPI IEPs did not discriminate among G. rostochiensis standard pathotypes. Subsequently, 14 field populations of G. rostochiensis, five from Italy and nine from Venezuela, and three field populations of G. pallida, two from Italy and one from Chile, were assayed to obtain SOD IEPs. Italian populations had previously been identified at pathotype level by bioassays according to the generally accepted international test using different resistant potato cultivars and clones. The cluster analysis carried out on the SOD IEPs of all the populations tested formed four distinct groups within G. rostochiensis and only one within G. pallida. Pathotype identification of Globodera populations by SOD IEPs was not able to discriminate between bioassay standard couples Ro2/Ro3, Ro4/Ro5 and Pa2/Pa3. Therefore, three groups were assigned to Ro1, Ro2/3 and Ro4/5, and a fourth group to Pa2/Pa3. Four Venezuelan populations, not identified at pathotype level by bioassays, formed a distinct fifth group. By means of the method described herein, four additional unknown Venezuelan populations could be assigned to Ro1 group and one to Ro2/Ro3 group; one G. pallida population from Chile was assigned to Pa2/Pa3 group.

Antioxidant enzymes in (a)virulent populations of root-knot nematodes

Assays of antioxidant enzymes, including catalase, peroxidase and superoxide dismutase (SOD), have been carried out on extracts of females and second-stage juveniles (J2) of a pair of Meloidogyne incognita isolates, one virulent and one avirulent, selected on tomato, and an avirulent field population of M. incognita. Catalase and SOD activity were found to be higher in extracts of the virulent isolate SM1 when compared with the avirulent counterparts. Peroxidase activity, assayed with o-dianisidine as the substrate, was enhanced in SM1 J2 compared with the avirulent avr1 J2. Catalase isozymes were separated by isoelectrofocusing into a very acidic and a basic isoform; this latter isoform was found to be responsible for the enhancement of catalase activity in virulent populations. SOD isozyme electrophoresis patterns (IEP) of root-knot nematodes, obtained by native PAGE, showed the presence of slow- and fast-migrating bands. SOD IEP of virulent females contained a slow-migrating band with a relative mobility (Rm) on the gels slightly higher (0.52) than the corresponding band from avirulent populations (0.50). This change was confirmed with native PAGE gels loaded with extracts from J2. To check how widespread this change is in field populations of RKN, a survey of SOD IEP from 12 RKN field (a)virulent populations was carried out. The specificity of the 0.52 Rm band for virulent populations was confirmed. Separation by native PAGE of peroxidases, stained either by o-dianisidine or diamino-benzidine, showed two isoforms with no apparent differences between the populations tested.