A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

AIMS: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. METHODS AND RESULTS: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. CONCLUSIONS: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. SIGNIFICANCE AND IMPACT OF THE STUDY: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Virulence Genes in Heterodera glycines: Allele Frequencies and Ror Gene Groups Among Field Isolates and Inbred Lines.

ABSTRACT Genetic variation in field populations of Heterodera glycines is a key issue for both resistance gene deployment and basic understanding of virulence-gene flow in populations. In this study, we examined phenotypically defined genes for virulence under selection from host resistance. We separated the most common H. glycines genotypes in the United States into two virulence groups, based on their reproductive abilities on the resistant soybean plant introduction (PI) 88788. These groups correspond to previously identified virulence genes in the nematode, as follows: the dominant gene in H. glycines to PI88788, and the recessive genes to PI90763 and Pickett/Peking. Virulence allele frequencies and virulence genotype frequencies of selected field isolates were investigated by testing the host range of single-female-derived lines, which were developed through single-female inoculation on the standard susceptible soybean 'Lee 68'. By comparing virulence genotype frequencies between the original field isolates and their single-female-derived lines, we were able to determine allele frequencies in the field populations. The results suggest that tremendous variation in H. glycines virulence genes exists among field populations. Potential mechanisms of selection which could cause virulence genotype frequency increases are discussed as related to population genetics equilibrium theory.

A Revised Classification Scheme for Genetically Diverse Populations of Heterodera glycines.

Heterodera glycines, the soybean cyst nematode, is a major yield-limiting pathogen in most soybean production areas worldwide. Field populations of H. glycines exhibit diversity in their ability to develop on resistant soybean cultivars. Since 1970, this diversity has been characterized by a bioassay used to assign a race classification to a population. The value of the race scheme is reflected in the number and quality of resistant soybean cultivars that have been developed and released by soybean breeders and nematologists working in concert. However, the race scheme also has been misapplied as a means of studying H. glycines genotypes, in part due to the use of the term "race." For fungal and bacterial pathogen species, "race" can theoretically be applied to individuals of a population, thus allowing inference of individual genotypes. Application of a race designation to an individual egg or second-stage juvenile (J2) of H. glycines is not possible because a single J2 cannot be tested on multiple hosts. For other nematode species, "race" is defined by host ranges involving different plant species, whereas the H. glycines race test involves a set of lines of the same plant species. Nonetheless, because H. glycines populations vary in genetic diversity, and this variation has implications for management strategies, a mechanism is needed for documenting and discussing population differences. The HG Type scheme described herein avoids the implication of genetic uniformity or predictability in contrast to the way the race scheme has been used.

Reproductive Strategies and Karyotype of the Burrowing Nematode, Radopholus similis.

Karyotype, gametogenesis, and gonad morphology were characterized for 56 Radopholus spp. isolates collected from Africa, Australia, Central America, Cuba, Dominican Republic, Guadeloupe, Puerto Rico, North America (Florida), and Hawaii. Seven of the isolates, all collected from Florida, were citrus-parasitic. The haploid karyotype for all isolates was n = 5, and gonad organization was similar for each. Furthermore, reproduction did not involve parthenogenesis. Initially, spermatids were produced in young adult females and accumulated in the spermatheca prior to differentiation to sperm. At the cessation of spermatogenesis, oogenesis began and continued for the remainder of the nematode's life. Oocytes first entered a mitotic phase, then a transition zone, and remained in pachytene until they reached the proximal end of the ovary. Thus, Radopholus can reproduce as a hermaphrodite when amphigony does not occur. The gonad is actually an ovatestis.

Phylogenetic Analysis of Geographically Diverse Radopholus similis via rDNA Sequence Reveals a Monomorphic Motif.

The nucleic acid sequences of rDNA ITS1 and the rDNA D2/D3 expansion segment were compared for 57 burrowing nematode isolates collected from Australia, Cameroon, Central America, Cuba, Dominican Republic, Florida, Guadeloupe, Hawaii, Nigeria, Honduras, Indonesia, Ivory Coast, Puerto Rico, South Africa, and Uganda. Of the 57 isolates, 55 were morphologically similar to Radopholus similis and seven were citrus-parasitic. The nucleic acid sequences for PCR-amplified ITS1 and for the D2/D3 expansion segment of the 28S rDNA gene were each identical for all putative R. similis. Sequence divergence for both the ITS1 and the D2/D3 was concordant with morphological differences that distinguish R. similis from other burrowing nematode species. This result substantiates previous observations that the R. similis genome is highly conserved across geographic regions. Autapomorphies that would delimit phylogenetic lineages of non-citrus-parasitic R. similis from those that parasitize citrus were not observed. The data presented herein support the concept that R. similis is comprised of two pathotypes-one that parasitizes citrus and one that does not.

Randomly Amplified Polymorphic DNA Differs with Burrowing Nematode Collection Site, but not with Host Range.

The genetic variability of 12 burrowing nematode (Radopholus sp.) isolates from Central America, the Caribbean, and Florida, and one isolate from Ivory Coast were compared with RAPD analysis. A high degree of genetic similarity (>0.82) was determined for isolates from the Western Hemisphere. Genome similarity was greatest among isolates collected within a country. Among isolates collected in Central America and the Caribbean, burrowing nematodes from Belize and Guatemala were genetically more distant. However, the genome of the isolate from Ivory Coast was most dissimilar (>0.30). These results suggest that African and American burrowing-nematode isolates may have had different origins or that they have been geographically isolated for a sufficient amount of time to have accumulated genetic changes detectable by RAPD analysis. No relationship was found between the genomic similarity and extent of reproduction or damage to banana or citrus roots. Morphometric analysis involving eight of the isolates indicated that they were morphologically identical and values for morphometric parameters were well within the range previously published for banana and citrusparasitic burrowing nematodes.

Aggressiveness and Damage Potential of Central American and Caribbean Populations of Radopholus spp. in Banana.

Monoxenic cultures of burrowing nematode populations extracted from banana roots from Belize, Guatemala, Honduras, and Costa Rica were established on carrot discs. Cultures of Radopholus spp. were also obtained from Florida, Puerto Rico, Dominican Republic, and Ivory Coast. The aggressiveness (defined as reproductive fitness and root necrosis) of these populations was evaluated by inoculating banana plants (Musa AAA, cv. Grande Naine) with 200 nematodes/plant. Banana plants produced by tissue culture were grown in 0.4-liter styrofoam cups, containing a 1:1 mix of a coarse and a fine sand, at ca. 27 degrees C and 80% RH. Banana plants were acclimated and allowed to grow for 4 weeks prior to inoculation. Plant height, fresh shoot and root weights, root necrosis, and nematode population densities were determined 8 weeks after inoculation. Burrowing-nematode populations varied in aggressiveness, and their reproductive fitness was generally related to damage reported in the field. Plant height and fresh shoot and root weight did not reflect damage caused by nematodes under our experimental conditions. Necrosis of primary roots was closely related to the reproductive fitness of the nematode populations. Variation in aggressiveness among nematode populations followed a similar trend in the two susceptible hosts tested, Grande Naine and Pisang mas. All nematode populations had a low reproductive factor (Rf

THE CAENORHABDITIS ELEGANS GENOME: A Guide in The Post Genomics Age.

The completion of the entire genome sequence of the free-living nematode, Caenorhabditis elegans is a tremendous milestone in modern biology. Not only will scientists be poring over data mined from this resource, but techniques and methodologies developed along the way have changed the way we can approach biological questions. The completion of the C. elegans genomic sequence will be of particular importance to scientists working on parasitic nematodes. In many cases, these nematode species present intractable challenges to those interested in their biology and genetics. The data already compared from parasites to the C. elegans database reveals a wealth of opportunities for parasite biologists. It is likely that many of the same genes will be present in parasites and that these genes will have similar functions. Additional information regarding differences between free-living and parasitic species will provide insight into the evolution and nature of parasitism. Finally, genetic and genomic approaches to the study of parasitic nematodes now have a clearly marked path to follow.

The soybean cyst nematode, Heterodera glycines: a genetic model system for the study of plant-parasitic nematodes.

Despite advances in understanding plant responses to nematode infection, little information exists regarding parasitic mechanisms. Recently, it has become possible to perform genetic analysis of soybean cyst nematode. Integration of classic and reverse genetics and genomic approaches for the parasite, with host genetics and genomics will expand our knowledge of nematode parasitism.

Caenorhabditis elegans: A Genetic Guide to Parasitic Nematode Biology.

The advent of parasite genome sequencing projects, as well as an increase in biology-directed gene discovery, promises to reveal genes encoding many of the key molecules required for nematode-host interactions. However, distinguishing parasitism genes from those merely required for nematode viability remains a substantial challenge. Although this will ultimately require a functional test in the host or parasite, the free-living nematode Caenorhabditis elegans can be exploited as a heterologous system to determine function of candidate parasitism genes. Studies of C. elegans also have revealed genetic networks, such as the dauer pathway, that may also be important adaptations for parasitism. As a more directed means of identifying parasitism traits, we developed classical genetics for Heterodera glycines and have used this approach to map genes conferring host resistance-breaking phenotypes. It is likely that the C. elegans and H. glycines genomes will be at least partially syntenic, thus permitting predictive physical mapping of H. glycines genes of interest.

Genetic analysis of parasitism in the soybean cyst nematode Heterodera glycines.

A genetic analysis of parasitic ability in the soybean cyst nematode Heterodera glycines was performed. To identify and characterize genes involved in parasitism, we developed three highly inbred H. glycines lines, OP20, OP25 and OP50, for use as parents for controlled crosses. Through these crosses, we have identified genes in the inbred parents that control reproduction of the nematode on hosts that carry resistance genes. These genes, designated as ror-* for reproduction on a resistant host, segregate in a normal Mendelian fashion as independent loci. Host range tests of F1 generation progeny indicated that at least one parasitism gene in both the OP20 and OP50 lines for host PI 88788 was dominant. Parasitism genes in OP50 for hosts "Peking" and PI 90763 are recessive. Two types of single female descent populations, a single backcrossed BC1F2-derived and a double backcrossed BC2F1-derived, were established on the susceptible soybean cultivar "Lee 68." Host range tests for parasitism in these lines demonstrated the presence of two independent genes in OP50, one for host PI 88788 designated ror-1 and one for host PI 90763 designated ror-2. OP20 carries two independent genes for parasitism on PI 88788, designated as alleles kr3 and kr4.

Sequence Tag Site and Host Range Assays Demonstrate that Radapholus similis and R. citraphilus are not Reproductively Isolated.

Males of citrus-parasitic Radopholus citrophilus (FL1) were mated with non-citrus-parasitic R. similis (FL5) females. Progeny inherited a 2.4-kb sequence tag site (DK#1) and the ability to reproduce in citrus from the paternal parent (FLl); both traits were absent in the maternal line (FL5). The hybrid progeny produced offspring in roots of citrus seedlings over an 8-month period and therefore were considered reproductively viable. Genomic DNA hybridization studies indicated that one or more copies of DK#1 were present in R. citrophilus FL1. It is not likely that DK#1 represents a citrus parasitism gene because it was amplified from some burrowing nematode isolates that did not parasitize citrus and because DK#1 contains no open reading frames. Inability to reliably test individual nematodes for their ability to parasitize citrus was a constraint to obtaining F2 data required for definitive genetic characterization of citrus parasitism in burrowing nematodes, and alternate approaches will be required. Although the physical relationship of DK#1 and the citrus parasitism locus remains undefined, results of controlled mating studies using these parameters as genetic markers enabled us to identify hybrid F progeny. Therefore, R. similis and R. citrophilus are not sibling species since gene flow between the two does not appear to be restricted via geographic isolation (sympatric in Florida) or by genetics.

Genome Similarity Implies that Citrus-Parasitic Burrowing Nematodes do not Represent a Unique Species.

Burrowing nematodes from Central America, Dominican Republic, Florida, Guadeloupe, Hawaii, and Puerto Rico were characterized for their ability to parasitize citrus, but citrus parasites were found only in Florida. Sequence tag sites originally amplified from a citrus-parasitic burrowing nematode were polymorphic among 37 burrowing nematode isolates and were not correlated with citrus parasitism, nematode isolate collection site, or amplification of a 2.4-kb sequence tag site (DK#1). Results of a RAPD analysis and characterization of the isozymes phosphoglucose isomerase, lactate dehydrogenase, and malate dehydrogenase indicated that the burrowing nematode isolates were highly similar. Citrus parasitism in Florida appears to be associated with limited changes in the burrowing nematode genome. Findings did not substantiate a previous report that R. citrophilus was present in Hawaii. Overall, these data do not support assignment of sibling species status to burrowing nematodes that differ with respect to citrus parasitism.

Genetics of Soybean-Heterodera glycines Interactions.

The soybean cyst nematode, Heterodera glycines, is one of the most economically important pathogens of soybean. Effective management of the nematode is often dependent on the planting of resistant soybean cultivars. During the past 40 years, more than 60 soybean genotypes and plant introductions (PI) have been reported as resistant to H. glycines. About 130 modern soybean cultivars registered in the United States are resistant to certain races of H. glycines. Several resistance genes have been identified and genetically mapped; however, resistance levels in many soybean cultivars are not durable. Some older cultivars are no longer resistant to certain H. glycines populations in many production areas, especially if a soybean monoculture has been practiced. Past soybean registration reports show that all resistant cultivars developed in public institutions from the mid-1960s to the present have been derived from five PIs. This narrow genetic background is fragile. To further complicate the issue, soybean-H. glycines genetic interactions are complex and poorly understood. Studies to identify soybean resistance genes sometimes have overlapped, and the same genes may have been reported several times and designated by different names. Nevertheless, many potential resistance genes in existing germplasm resources have not yet been characterized. Clearly, it is necessary to identify new resistance genes, develop more precise selection methods, and integrate these resistance genes into new cultivars. Rational deployment of resistant cultivars is critical to future sustained soybean production.

Molecular polymorphisms associated with host range in the highly conserved genomes of burrowing nematodes, Radopholus spp.

Six polymorphic bands of DNA were amplified from purified Radopholus citrophilus genomic DNA from one strain of each of the sibling species R. citrophilus and R. similis in random amplified polymorphic DNA analyses involving 380 single 10-base primers. Four of these polymorphic DNA fragments were successfully cloned and amplified through subsequent use of primers designed to complement the terminal sequences of the polymorphic DNA. Results of ensuing studies using mini-prepped DNA from 14 burrowing nematode strains collected from Florida, Hawaii, and Central America, characterized for their ability to parasitize citrus, indicated that a 2.4-kb fragment appeared to be associated with citrus parasitism in burrowing nematode populations from Florida. However, a fragment of comparable size was also detected in R. citrophilus from Hawaii and from burrowing nematode populations collected from Belize and Puerto Rico. Overall, findings suggest that the genome organization of the burrowing nematode sibling species R. citrophilus and R. similis is highly conserved. This remarkable genetic similarity should facilitate identification of genetic sequence related to important phenotypes such as citrus parasitism. Detection of R. citrophilus-specific DNA fragments in burrowing nematodes collected from Belize and Puerto Rico suggests that R. citrophilus is resident in some Central American countries.

Root-knot nematode--directed expression of a plant root--specific gene.

Root-knot nematodes are obligate plant parasites that induce development of an elaborate feeding site during root infection. Feeding-site formation results from a complex interaction between the pathogen and the host plant in which the nematode alters patterns of plant gene expression within the cells destined to become the feeding site. Expression of TobRB7, a gene expressed only in tobacco roots, is induced during feeding site development. The cis-acting sequences that mediate induction by the nematode are separate from those that control normal root-specific expression. Reporter transgenes driven by the nematode-responsive promoter sequences exhibit expression exclusively in the developing feeding site.

Nematode acetylcholinesterases: molecular forms and their potential role in nematode behavior.

Nematode movement is reliant upon the somatic musculature that runs longitudinally along the body wall. Neuromuscular synapses occur in the ventral and dorsal cords and employ the excitatory neurotransmitter, acetylcholine (ACh), for modulation of muscle activity. Acetylcholine activity is terminated by hydrolysis by acetylcholinesterase (AChE). Here, Charles Opperman and Stella Chang discuss the molecular forms and potential role of this enzyme.

Separation and Characterization of Heterodera glycines Acetylcholinesterase Molecular Forms.

The composition and biochemical properties of acetylcholinesterases isolated from Heterodera glycines were determined. Heterodera glycines contains three separable AChE molecular forms that can be grouped into two classes corresponding to classes A and C found in some other nematode species. The apparent lack of class B AChE is unusual and may have significant behavioral ramifications. The class C enzyme isolated from H. glycines is similar to that from Meloidogyne arenaria and M. incognita but is somewhat more sensitive to AChE inhibitors such as eserine. Heterodera glycines possesses a larger percentage of its total acetylcholinesterase as class C than other nematodes thus far examined.

Characterization of acetylcholinesterase molecular forms of the root-knot nematode, Meloidogyne.

Multiple molecular forms of acetylcholinesterase have been isolated and characterized from the root-knot nematodes Meloidogyne arenaria and Meloidogyne incognita. The forms of enzyme present in these 2 species are similar but not identical to those that occur in the free-living nematode Caenorhabditis elegans. The 5 enzyme forms exhibit differential solubilities and can be classified into 3 classes, A, B, and C, based on substrate affinity, inhibitor and detergent sensitivity, and thermal inactivation profiles. An unusual class of acetylcholinesterase has been isolated from Meloidogyne which has very high affinity for acetylcholine, but is highly resistant to carbamate and organophosphate inhibitors. The potential roles of the molecular forms in nematode behavior and sensitivity to nematicides are discussed.