Cultivation of monoxenous trypanosomatids: A minireview.

Trypanosomatids are obligatory parasites, some of which are responsible for important human and animal diseases, but the vast majority of trypanosomatids are restricted to invertebrate hosts. Isolation and in vitro cultivation of trypanosomatids from insect hosts enable their description, characterization, and subsequently genetic and genomic studies. However, exact nutritional requirements are still unknown for most trypanosomatids and thus very few defined media are available. This mini review provides information about the role of different ingredients, recommendations and advice on essential supplements and important physicochemical parameters of culture media with the aim of facilitating first attempts to cultivate insect-infesting trypanosomatids, with a focus on monoxenous trypanosomatids.

Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.

AIMS: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus more costly and time-consuming, or using only one fluorescent dye, thus less accurate. METHODS AND RESULTS: A red primary fluorescence (autofluorescence) of the hyphal cell walls and septa of Rhizoctonia spp. with green excitation is evidenced in Rhizoctonia spp. This property is exploited and combined for the first time with a conventional DAPI fluorescence to accurately determine the nuclear condition of Rhizoctonia. This bi-fluorescence imaging strategy depicted the nuclear condition in Rhizoctonia spp. more accurately than the conventional DAPI fluorescence used alone and was validated against isolates previously genotyped by DNA sequencing. CONCLUSIONS: We demonstrated that the bi-fluorescence imaging strategy was safe, accurate and simple to perform and interpret. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed bi-fluorescence imaging strategy provides a sensitive tool for determining the nuclear condition of Rhizoctonia strains. Its simplicity is a key advantage when there are numerous cultures to be examined.

First Report of Black Swallow-Wort as an Alternate Host of the Two-Needle Pine Stem Rust Pathogen, Cronartium flaccidum, in France.

Stem rust disease, caused by Cronartium flaccidum (Alb. & Schwein.) G. Winter, is among the most destructive diseases of the two-needle hard pine in the Northern Hemisphere, including Scots pine but also several Mediterranean pines in southern Europe (2,3). This heteroecious rust has numerous alternate herbaceous hosts spanning different plant families, thereby contributing to epidemic outbreaks when environmental conditions for infection are optimal (2,3). The main alternate host in Europe is the white swallow-wort, Vincetoxicum hirundinaria Medik, a herbaceous perennial in the milkweed family (Asclepiadaceae). At the southwestern edge of its distribution, V. hirundinaria co-occurs with the black swallow-wort, V. nigrum (L.) Moench and cases of misidentification between the two species are not uncommon. Little to no disease occurs to V. nigrum likely because phenanthroindolizidine alkaloid antimicrobial compounds are produced in the weed. In 1918, occurrence of C. flaccidum was reported in Spain and Portugal on black and white swallow-worts albeit as C. asclepadium (1). In the early summer of 2011, at Saint Clément de Rivière in southern France, we detected orange-yellow rust pustules on the lower leaf surfaces of several black swallow-worts growing near Aleppo pines (Pinus halepensis). These orange-yellow pustules were erumpent uredinia in groups (range = 137 to 400 µm in diameter) with peridia that broke with the production of uredinospores. The latter were moderately echinulate, light yellow, broadly ellipsoid (length = 23 ± 4 µm and range = 11 to 33 µm; width = 15 ± 2 µm and range = 9 to 20 µm) with walls of 1 to 2 µm thick (mean 1.3 ± 0.2 µm). Hair-like columnar telia (length = 1123 ± 131 µm and range = 976 to 1280 µm; width = 136 ± 28 µm and range = 104 to 176 µm) were mostly formed from uredinia. Telia were hypophyllous and reddish orange. Teliospores were orange-yellow and ellipsoidal to cylindrical (length 26.3 ± 6.2 µm and range 13.5 to 46 µm; width = 10.5 ± 1.8 µm and range = 6.9 to 14.9 µm). Morphological features of these fruiting structures were consistent with those of C. flaccidum (Alb. & Schwein.) G. Winter on white swallow-worts (2). Additional confirmation was provided by sequencing the two internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S gene (4). The sequence was 843 bp long (GenBank Accession No. JN802139), 99.7% similar to C. flaccidum found on Melampyrum in Finland (Accession No. JF13709), and 99.4% similar to C. flaccidum found on pines in Italy (Accession No. X83900). Voucher material has been deposited at the Herbarium of Montpellier's University under the collection Accession No. MPUØ188846. To our knowledge, this is the first report of the occurrence of uredinia and telia of C. flaccidum on black swallow-worts clearly identified in France. The occurrence of the rust on this understory vine is of critical importance for the economic sustainability of pine forests in France, especially when they are heavily constrained by drought and fire. References: (1) R. Gonzalez Fragoso. Trab. Mus. Nac. Ci. Nat., Ser. Bot. 15:1, 1918. (2) J. Kaitera and H. Nuorteva. For. Pathol. 33:205, 2003. (3) A. Ragazzi. Phytopathol. Medit. 28:5, 1989. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Identification of 21 polymorphic microsatellites in the African parasitoid wasp, Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae).

We have developed 21 dinucleotide repeat microsatellite loci from African populations of Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae), a parasitoid wasp of the olive fruit fly, as part of a study assessing the role of introgression/hybridization in the success of a biological control introduction. We proposed suitable conditions for polymerase chain reaction multiplexing. All 21 loci were polymorphic with two to 21 alleles per locus within the Kenyan and South African populations tested. Most of them were successfully amplified in two other Psyttalia species.

High through-put characterization of insect morphocryptic entities by a non-invasive method using direct-PCR of fecal DNA.

The development of non-invasive molecular techniques is currently increasing, particularly in the fields of behavioural ecology and conservation genetics of mammals. Surprisingly, genetic studies of Arthropods and particularly the insects have not benefited yet from the contributions that non-invasive methods have made. Here, we outline a strategy for identifying phytophagous insect genetic entities based on direct-PCR of fecal DNA combined with double strand conformation polymorphism (DSCP) typing. This allows the differentiation of morphocryptic entities within the species Ceutorhynchus assimilis (Coleoptera: Curculionidae), a candidate biocontrol agent of a noxious weed. The results obtained clearly demonstrate the potential for this method to provide a valuable means for genetic and ecological studies of Arthropods.

Influence of temperature preferences of two Paecilomyces fumosoroseus lineages on their co-infection pattern.

In order to clarify the epidemiological potential of entomopathogenic fungi for insect pest control, the role of the temperature as one environmental constraint was investigated on the pattern of co-infection of Galleria mellonella by two distinct lineages of a hyphomycete, Paecilomyces fumosoroseus. The distribution of conidial populations collected on cadavers of hosts co-infected under 20 regimes, ranging from 13 to 35 degrees C, was examined. The apparent temperature tolerance of both fungal isolates was related to their in vitro colony growth and their in vivo sporulation ability. The conidial populations were characterized by molecular markers based on restriction fragment length polymorphisms of the internal transcribed spacers (ITS-RFLP) and random amplified polymorphic DNA (RAPD) contrasting profiles in combination with the conidial size. This study allowed a different temperature profile was identified for each isolate. Under most temperature regimes, only one lineage prevailed upon the infected insect; whereas both lineages coexisted at 20-25 and 25-25 degrees C. When one haplotype dominated, the displacement of the other one depended on its temperature tolerance. These results suggest that more consideration should be given to population-genetics analyses for evaluating the adaptability of microbial control agents to targeted environments.

Isozymes of a rattan species and their genetic interpretation.

A combination of a modified Feret' (Silvae Genet. 1971, 20, 46-50) extraction buffer and two types of electrophoresis with acrylamide and starch gels were used to characterize allozymes in mature vegetative tissue of a commercially high value species of rattans (Calamus subinermis). From the analysis of allelic segregation from single maternal rattans and their offspring, genetic control of the 16 observed banding zones, which were consistently scorable, was assumed. Seventeen gene loci were identified. The percentage of polymorphic loci within Calamus subinermis was much higher (70.5%) than expected levels of genetic diversity for tropical woody and non-woody species. It is thought that the protocol described may be applied to the analysis of the genetic diversity of all the endangered Calamus species.

A two-dimensional electrophoresis procedure for single meristems of different forest species.

High resolution two-dimensional electrophoresis, with isoelectric focusing in the first dimension and electrophoresis in sodium dodecyl sulfate in thin acrylamide gels in the second dimension, has been applied to separate the proteins of single meristems (200-300 microns) from Sequoiadendron giganteum, Sequoia sempervirens, Pseudotsuga menziesii, Picea abies, Pinus pinaster, Eucalyptus gunnii and Populus nigra. The technique may prove an efficient tool for studying inter- and intraclonal differences in meristem cultures of forest species, especially when referring to cloning from meristems of selected trees in relation to the rejuvenation process.

J 16: An apex protein associated with juvenility of Sequoiadendron giganteum.

Shoot apex proteins from 11 seedlings, two juvenile clones and three mature grafted clones of Sequoiadendron giganteum (Lindl.) Buchh. were examined throughout the year by SDS-polyacrylamide gel electrophoresis. A membrane-associated protein known as J 16, with an approximate molecular weight of 16 kD, was always detected in juvenile tissue, but was absent in tissue of the mature clones. The J 16 protein was also present in extracts of a mature clone that had been morphologically rejuvenated by micrografting. Protein extracts from the rejuvenated clone, were separated by electrophoresis and assayed for J 16 using an immunoblotting procedure with anti-J 16 serum. This test revealed two bands, J 16 and another protein with a lower molecular weight. The small molecular weight protein was not detected in juvenile clones.