Phenylethanoid glycosides from Lippia javanica

Lippia javanica (N.L.Burm.) Spreng. is an aromatic, multipurpose medicinal plant from which a number of volatile compounds have been identified, together with toxic triterpenoids and iridoid glycosides. Two additional phenylethanoid glycosides, verbascoside and isoverbascoside, were isolated from L. javanica and characterized. High performance liquid chromatography analyses of polar extracts of three other Lippia species (L. scaberrima, L. rehmannii and L. wilmsii), indigenous to South Africa, revealed the presence of both isomers. When compared to the other indigenous Lippia species, the leaves of L. javanica were found to contain the highest concentrations of both isomers. In addition, the intraspecies variation of the verbascoside/isoverbascoside content of L. javanica, harvested from the same and different localities, was investigated. The concentrations of the two phenylethanoids remained fairly consistent within and between different populations, even when geographically separated. While these compounds are produced by many genera, they may now be added to the list of iridoid glucosides employed as chemotaxonomic markers for Lippia species.     

A chemotaxonomic assessment of four indigenous South African Lippia species using GC–MS and vibrational spectroscopy of the essential oils

Lippia species are widely used as traditional remedies by many South African communities. The identification of Lippia spp. indigenous to South Africa is currently based on the morphology and geographical origin of the specimen. However, morphological similarities are evident and due to sympatric distributions, different species may co-occur in the same natural environment, thereby presenting difficulties in the taxonomic delimitation of these taxa. In this paper, gas chromatography–mass spectrometry (GC–MS) and vibrational spectroscopic analyses were used to investigate the chemotaxonomic differentiation of four Lippia species (L. javanica, L. scaberrima, L. rehmannii and L. wilmsii), based on the essential oil composition. The results demonstrate that multivariate classification of chromatographic data enabled separation of Lippia oils into three distinct clusters representing L. scaberrima, L. rehmannii and L. javanica. With the aid of chemometric algorithms, it was also possible to use both mid infrared (MIR) and near infrared (NIR) spectroscopy for a clear distinction of the Lippia spp., with results comparable to those obtained by GC–MS. Orthogonal projections to latent structures (OPLS) presented a better classification algorithm than principal component analysis (PCA), in both GC–MS and spectroscopy analyses. Chemical variations within the Lippia spp. complex were evident, particularly for L. javanica and L. scaberrima, which exhibited significant intra-species variation.     

Biology and host range of <Emphasis Type="Italic">Aconophora compressa,</Emphasis> a candidate considered as a biocontrol agent of <Emphasis Type="Italic">Lantana camara</Emphasis> in Africa

The shrub, Lantana camara L. (Verbenaceae), of tropical American origin, is a serious weed in South Africa and is the target of a biological control programme. The stem-sucking membracid, Aconophora compressa Walker (Homoptera: Membracidae), from Mexico was reported to be very damaging and was therefore imported into South Africa for biology and host range studies. The female partially inserts the eggs into the woody portion of actively growing stems, and guards them against potential predators. Nymphs develop through five instars to the adult stage in about 45 days. The adults and nymphs feed on the sap of stems causing the leaves to wilt, the flowers to abort and the gradual dieback of stems. No-choice experiments showed that adult survival, egg production, and nymphal emergence was high on L. camara, and also on related ornamental and indigenous species. The adult reproductive performance was higher on some indigenous Lippia species than on L. camara. Furthermore, the nymphs developed faster on these indigenous species and high rates of reproductive performance on these non-target species were sustained over several generations. In multiple-choice trials, equal or larger numbers of egg batches were recorded from indigenous Lippia species and the ornamental plant, Aloysia citrodora Palau, than on L. camara. The treehopper, A. compressa, poses an unacceptable threat to indigenous Lippia species and has therefore been rejected as a biocontrol agent for L. camara in Africa.     

Chemical composition and antifungal activity of the essential oils of Lippia rehmannii from South Africa

Lippia rehmannii H.Pearson (Verbenaceae) is an aromatic bush, indigenous to the northern parts of South Africa. As far as could be ascertained, the essential oil composition has not been previously reported and forms the subject of this investigation. Aerial parts of the shrub were collected from two localities in Gauteng, South Africa, and the isolated essential oils were analysed by gas chromatography. Citral, a mixture of the E- and Z-isomers, was found to be the main constituent of the oils, while borneol, camphor, neryl acetate, isocaryophyllene, p-cymene, β-caryophyllene and β-caryophyllene oxide were other major compounds present. Oil compositions, within and between the two localities, did not differ significantly. The in vitro antifungal activity of L. rehmannii essential oil was compared to that of Cympopogon citratus (lemongrass) and pure citral, against a number of pre- and postharvest fungal food pathogens. At a concentration of 3000 µL/L, lemongrass oil and pure citral caused complete growth inhibition of all the pathogens tested. Lippia rehmannii, containing less citral than lemongrass oil, was effective at this concentration against the majority of pathogens, but only partially restricted the growth of Lasiodiplodia theobromae and Botrytis cinerea. This finding suggests that citral may be largely responsible for the observed antifungal activities. Essential oil from L. rehmannii appears to be a good candidate for the in vitro control of Fusarium oxysporum and Rhizoctonia solani and application of these oils in the field should be investigated.     

Identification of Single Meloidogyne Juveniles by Polymerase Chain Reaction Amplification of Mitochondrial DNA

Polymerase chain reaction (PCR) was used to amplify a specific 1.8-kb sequence of mitochondrial DNA from single juveniles and eggs from 17 populations of Meloidogyne incognita, M. hapla, M. javanica, and M. arenaria. Approximately 2 μg amplified product were produced per reaction. Restriction digestion of the amplified product with HinfI permitted discrimination of clonal lineages of the four species. Meloidogyne javanica, however, could not be separated from M. hapla by the enzymes used in these experiments. Various amplification conditions and nematode lysis procedures were examined in order to optimize the speed and quality of identifications.     

Damage Functions for Three Meloidogyne Species on Arachis hypogaea in Texas

The yield response of Florunner peanut to different initial population (Pi) densities of Meloidogyne arenaria, M. javanica, and an undescribed Meloidogyne species (isolate 93-13a) was determined in microplots in 1995 and 1996. Seven Pi''s (0, 0.5, 1, 5, 10, 50, and 100 eggs and J2/500 cm³ soil) were used for each Meloidogyne species in both years. The three species reproduced abundantly on Florunner in both years. In 1995, mean reproduction differed among the three species; mean Rf values were 10,253 for isolate 93-13, 4,256 for M. arenaria, and 513 for M. javanica. In 1996, the reproduction of M. arenaria (mean Rf = 7,820) and isolate 93-13a (mean Rf = 7,506) were similar, and both had greater reproduction on peanut than did M. javanica (mean Rf = 2,325). All three nematode species caused root and pod galling, and a positive relationship was observed between Pi and the percentage of pods galled. Meloidogyne arenaria caused a higher percentage of pod galling than did M. javanica or isolate 93-13a. A negative linear relationship between log₁₀ (Pi + 1) and pod yield was observed for all three nematode species each year. The yield response slopes were similar except for that of M. javanica, which was less negative than that of isolate 93-13a in 1995, and less negative than that of M. arenaria and isolate 93-13a in 1996.     

Antioxidant status of Lobiger serradifalci and Oxynoe olivacea (Opisthobranchia, Mollusca)

Invasion of the Mediterranean Sea by the two world-wide famous exotic algae species, Caulerpa taxifolia and Caulerpa racemosa, is still a problem and has adverse effects on the Mediterranean sublittoral ecosystem. Biological control studies revealed that the two native Sacoglossans, Oxynoe olivacea and Lobiger serradifalci, may have an effect on the expansion of invasive Caulerpa spp. in the Mediterranean. In the framework of this study, antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, as oxidative stress markers in L. serradifalci and O. olivacea were determined at two different temperature conditions (20 and 27 °C). In both species, SOD, CAT and GSH-Px activities were found to be positively correlated with temperature. The SOD activities in L. serradifalci were higher than those in O. olivacea at both temperatures, whereas the CAT and GSH-Px activities were significantly (p<0.05) higher in O. olivacea, compared to L. serradifalci. As expected, both species showed decreased LPO levels at 27 °C compared to 20 °C. GSSG level at 27 °C in O.olivacea was significantly (p<0.05) higher than that of 20 °C. On the other hand, no statistical (p>0.05) difference in L.serradifalci existed between GSSG levels at two temperatures. But, despite the variations in the antioxidant enzyme activities, there was no significant difference in LPO levels between the species, suggesting that the oxidative consequences of a given environmental condition may vary among different species. Inasmuch as the GSSG levels were in accordance with antioxidant enzyme activities, GSH might have acted as a cofactor of GSH-Px and an individual antioxidant in these sea slugs.     

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.     

Larvicidal activity against Anopheles arabiensis of 10 South African plants that are traditionally used as mosquito repellents

Ethanol extracts of Aloe ferox, Atalaya alata, Balanites maughamii, Clausena anisata, Croton menyaarthii, Lippia javanica, Melia azedarach, Olax dissitiflora, Sclerocarya birrea and Trichilia emetica were evaluated for their larvicidal activity against Anopheles arabiensis mosquitoes. Larval mortality was observed after 24 h of exposure. Larvicidal activity was only found in 5 plant extracts, namely, C. anisata, C. menyaarthii, L. javanica, O. dissitiflora and T. emetica. The bark extract of O. dissitiflora exhibited the highest larvicidal activity with LC₅₀ value of 25.24 μg/ml. The results of the present study showed that the bark of O. dissitiflora may have the potential to be used as larvicides against An. arabiensis.     

Chromosome numbers in the genus Lippia (Verbenaceae)

The genus Lippia (Verbenaceae) comprises about 200 taxa mainly distributed in Brazil, Mexico, Central America, Africa, Argentina and Paraguay. Some problems involving the number and delimitation of species have been reported. In order to contribute to the solving of these problems, the chromosome numbers of 14 Lippia species are documented. The following species were collected at Espinhaço Range, Southeast Brazil: Section Zapania (L. corymbosa, L. diamantinensis, L. hermannioides, L. lacunosa, L. rotundifolia, L. rubella), section Rhodolippia (L. florida, L. lupulina, L. pseudothea, L. rosella), section Goniostlachyum (L. glandulosa, L. pohliana, L. sidoides) and section Dioicolippia (L.filifolia). Immature inflorescences were collected and the ideal size for chromosome observation was determined. The majority of species have a haploid chromosome number from 10 to 14. Few species have a higher chromosome number, which suggests the occurrence of polyploidy. The relationships between chromosome numbers and the taxonomic sections are also discussed.     

Effects of Rapeseed and Vetch as Green Manure Crops and Fallow on Nematodes and Soil-borne Pathogens

In a rapeseed-squash cropping system, Meloidogyne incognita race 1 and M. javanica did not enter, feed, or reproduce in roots of seven rapeseed cultivars. Both nematode species reproduced at low levels on roots of the third crop of rapeseed. Reproduction of M. incognita and M. javanica was high on squash following rapeseed, hairy vetch, and fallow. The application of fenamiphos suppressed (P = 0.05) root-gall indices on squash following rapeseed, hairy vetch, and fallow; and on Dwarf Essex and Cascade rapeseed, but not Bridger and Humus rapeseed in 1987. The incorporation of 30-61 mt/ha green biomass of rapeseed into the soil 6 months after planting did not affect the population densities of Criconemella ornata, M. incognita, M. javanica, Pythium spp., Rhizoctonia solani AG-4; nor did it consistently increase yield of squash. Hairy vetch supported larger numbers of M. incognita and M. javanica than rapeseed cultivars or fallow. Meloidogyne incognita and M. javanica survived in fallow plots in the absence of a host from October to May each year at a level sufficient to warrant the use of a nematicide to manage nematodes on the following susceptible crop.     

Comparative Studies on Root Invasion,Root Galling,and Fecundity of Three Meloidogyne spp. on a Susceptible Tobacco Cultivar

Root invasion, root galling, and fecundity of Meloidogyne javanica, M. arenaria, and M. incognita on tobacco was compared in greenhouse and controlled environment experiments. Significantly more M. javanica than M. arenaria or M. incognita larvae were found in tobacco roots at 2, 4, and 6 d after inoculation. Eight days after inoculation there were significantly more M. arenaria and M. javanica than M. incognita larvae. Ten days after inoculation no significant differences were found among the three Meloidogyne species inside the roots. Galls induced by a single larva or several larvae of M. javanica were significantly larger than galls induced by M. incognita: M. arenaria galls were intermediate in size. Only slight differences in numbers of egg masses or numbers of eggs produced by the three Meloidogyne species were observed up to 35 d after inoculation.     

Yield Response and Injury Levels of Meloidogyne incognita and M. javanica on the Susceptible Tobacco 'McNair 944'

The effects of Meloidogyne incognita and M. javanica on a susceptible tobacco (Nicotiana tabacum L.) cv. McNair 944 were investigated in field microplots during 1978 and 1979. Three initial inoculum levels—4, 16, and 64 nematode eggs and/or second-stage larvae per 100 cm³ of soil—were used for each nematode species. Data obtained from the experiments included plant yield and the amount of reproduction of the two nematode species. At comparative inoculum levels, M. javanica was more aggressive than M. incognita on tobacco and caused approximately twofold more yield suppression than M. incognita. The calculated initial population of M. incognita, derived from the average for 2 yr, which produced a 7% suppression in plant yield was four eggs and/or second-stage larvae per 100 cm³ of soil; whereas less than one M. javanica egg and/or second-stage larvae per 100 cm³ of soil was needed to achieve similar suppression. Nematode reproduction varied in the 1978 and 1979 tests, but similar trends were observed. Early season M. javanica populations were greater than those of M. incognita, but late season populations of M. incognita were twice anti three times those of M. javanica.     

Antimicrobial and antioxidant activities of Mexican oregano essential oils (Lippia graveolens H. B. K.) with different composition when microencapsulated inβ‐cyclodextrin

Aims: To study how the antimicrobial and antioxidant activities of Lippia graveolens essential oils with different composition are affected after the microencapsulation process with β‐cyclodextrin (βCD). Methods and results: Three Mexican oregano essential oils (EOs) with different carvacrol/thymol/p‐cymene ratios (38 : 3 : 32, 23 : 2 : 42, 7 : 19 : 35) were used in this study. Microencapsulation was carried out by spray‐drying. Antimicrobial activities were measured as MBC (minimal bactericidal concentration) using 0·05%/0·10%/0·20% (w/v) dilutions of EOs against Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538. Antioxidant activities were determined by the 2,2′‐diphenyl‐1‐picrylhydrazil (DPPH) method. EOs showed antimicrobial and antioxidant activity, but microencapsulation preserved the antimicrobial activity in all cases and increased the antioxidant activity from four‐ to eightfold. Conclusions: Although the Lippia essential oils were from the same species, their composition affects the biological activities before and after the microencapsulation process, as well as encapsulation efficiency. Our study supports the fact that microencapsulation of EOs in β‐cyclodextrin preserves the antimicrobial activity, improves the antioxidant activity and acts as a protection for EOs main compounds. Significance and Impact of the Study: Microencapsulation affects positively EOs main compounds, improves antioxidant activity and retains antimicrobial activity, enhancing the quality of the oils.     

Evaluation of several tree species for activity against the animal fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus causes severe problems in poultry production systems. Seven South African tree species were selected from the database of the Phytomedicine Programme based on its antifungal activity against the fungus Cryptococcus neoformans. The acetone leaf extracts of the selected species had minimum inhibitory concentrations (MICs) of 0.16 mg/ml and lower in the preliminary screening. The antibacterial and antifungal activities of hexane, dichloromethane, acetone and methanol extracts of the leaves were determined using a two-fold serial microdilution method against a range of commonly encountered animal pathogenic fungi (A. fumigatus, Candida albicans, C. neoformans, Microsporum canis and Sporothrix schenckii) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa). The plant species investigated were Combretum vendae (A.E. van Wyk) (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh.) Engl. (Anacardiaceae). All the extracts had activity against at least one of the test organisms over an incubation period of 24 or 48 h. The MIC values of the non-polar and intermediate polarity extracts of O. natalitia, K. anthotheca, C. vendae, C. harveyi, and P. longifolia had MICs as low as 0.08 mg/ml against at least one of the tested bacteria. Furthermore, the acetone extracts of L. alata, K. wilmsii, O. natalitia and C. vendae had antifungal activities with MIC values ranging from 0.04 to 0.08 mg/ml against at least one of the tested fungi. The average MIC values of the plant extracts against the different bacteria ranged from 0.17 to 2.11 mg/ml, while the range was 0.23–1.98 mg/ml for fungi. The Gram-positive bacteria (S. aureus and E. faecalis) were more susceptible to the plant extracts than the Gram-negative bacteria (E. coli and P. aeruginosa). E. faecalis was the most susceptible microbe and C. vendae extracts were the most active against nearly all the bacteria tested. The acetone extract of L. alata was the most active against fungal pathogens, with activity against at least 3 fungal organisms. L. alata was selected for further work to isolate compounds active against A. fumigatus and other fungal pathogens.     

Morphological Comparison of Head Shape and Stylet Morphology of Second-stage Juveniles of Meloidogyne Species

Head shape and stylet morphology of second-stage juveniles of one population each of M. incognita, M. javanica, M. arenaria, and M. hapla were compared by light microscopy. Excised stylets of each species were also compared by scanning electron microscopy (SEM). Differences in head morphology were observed only between M. hapla and the other three species. In SEM, differences in stylet size, shape, and relative distance of the dorsal esophageal gland orifice to the base of the stylet were evident. Differences in stylet morphology between M. incognita and M. javanica could not he detected by light microscopy, but M. arenaria and M. hapla could be distinguished from each other and from the other two species. Head shape and styler morphology of second-stage juveniles are considered useful taxonomic characters.     

Nucleotide Substitution Patterning within the Meloidogyne rDNA D3 Region and Its Evolutionary Implications

Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among acollection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified from genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in an isolate.     

Differential Response to Root-Knot Nematodes in Prunus Species and Correlative Genetic Implications

Responses of 17 Prunus rootstocks or accessions (11 from the subgenus Amygdalus and 6 from the subgenus Prunophora) were evaluated against 11 isolates of Meloidogyne spp. including one M. arenaria, four M. incognita, four M. javanica, one M. hispanica, and an unclassified population from Florida. Characterization of plant response to root-knot nematodes was based on a gall index rating. Numbers of females and juveniles plus eggs in the roots were determined for 10 of the rootstocks evaluated against one M. arenaria, one M. incognita, one M. javanica, and the Florida isolate. These 10 rootstocks plus Nemaguard and Nemared were retested by growing three different rootstock genotypes together in containers of soil infested individually with each of the above four isolates. Garfi and Garrigues almonds, GF.305 and Rutgers Red Leaf peaches, and the peach-almond GF.677 were susceptible to all isolates. Differences in resistance were detected among the other rootstocks of the subgenus Amygdalus. The peach-almond GF.557 and Summergrand peach were resistant to M. arenaria and M. incognita but susceptible to M. javanica and the Florida isolate. Nemaguard, Nemared, and its two hybrids G x N no. 15 and G x N no. 22 were resistant to all but the Florida isolate. In the subgenus Prunophora, Myrobalan plums P.1079, P.2175, P.2980, and P.2984; Marianna plum 29C; and P. insititia plum AD.101 were resistant to all isolates. Thus, two different genetic systems of RKN resistance were found in the subgenus Amygdalus: one system acting against M. arenaria and M. incognita, and another system also acting against M. javanica. Prunophora rootstocks bear a complete genetic system for resistance also acting against the Florida isolate. The hypotheses on the relationships between these systems and the corresponding putative genes of resistance are presented.     

A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

DNA Complexity of the Root-knot Nematode (Meloidogyne spp.) Genome

Cot curves derived from renaturation kinetics of sheared denatured DNA indicated that the genome of six populations representing the four most common root-knot nematode species (Meloidogyne incognita, M. arenaria, M. javanica, and M. hapla) is composed of 20% repetitive and 80% nonrepetitive sequences of DNA. Cot curves were almost identical, indicating that all populations had a haploid genome of approximately the same size. Calculations from an average Cot curve gave an estimate of 0.51 x 108 nucleotide base pairs for the haploid genome of the four Meloidogyne species. This genome is about 12-13 times larger than the genome of the E. coli strain used as a control.