Genetics and Mechanism of Resistance to Meloidogyne arenaria in Peanut Germplasm

Segregation of resistance to Meloidogyne arenaria in six BC₅F₂ peanut breeding populations was examined in greenhouse tests. Chi-square analysis indicated that segregation of resistance was consistent with resistance being conditioned by a single gene in three breeding populations (TP259-3, TP262-3, and TP271-2), whereas two resistance genes may be present in the breeding populations TP259-2, TP263-2, and TP268-3. Nematode development in clonally propagated lines of resistant individuals of TP262-3 and TP263-2 was compared to that of the susceptible cultivar Florunner. Juvenile nematodes readily penetrated roots of all peanut genotypes, but rate of development was slower (P = 0.05) in the resistant genotypes than in Florunner. Host cell necrosis indicative of a hypersensitive response was not consistently observed in resistant genotypes of either population. Three RFLP loci linked to resistance at distances of 4.2 to 11.0 centiMorgans were identified. Resistant and susceptible alleles for RFLP loci R2430E and R2545E were quite distinct and are useful for identifying individuals homozygous for resistance in segregating populations.     

Molecular Transfer of Nematode Resistance Genes

Recombinant DNA techniques have been used to introduce agronomically valuable traits, including resistance to viruses, herbicides, and insects, into crop plants. Introduction of these genes into plants frequently involves Agrobacterium-mediated gene transfer. The potential exists for applying this technology to nematode control by introducing genes conferring resistance to nematodes. Transferred genes could include those encoding products detrimental to nematode development or reproduction as well as cloned host resistance genes. Host genes that confer resistance to cyst or root-knot nematode species have been identified in many plants. The best characterized is Mi, a gene that confers resistance to root-knot nematodes in tomato. A map-based cloning approach is being used to isolate the gene. For development of a detailed map of the region of the genome surrounding Mi, DNA markers genetically linked to Mi have been identified and analyzed in tomato lines that have undergone a recombination event near Mi. The molecular map will be used to identify DNA corresponding to Mi. We estimate that a clone of Mi will be obtained in 2-5 years. An exciting prospect is that introduction of this gene will confer resistance in plant species without currently available sources of resistance.     

Genetics of Burley and Flue-Cured Tobacco Resistance to Globodera tabacum tabacum

Genotypes of burley (cultivars B-21 and B-49), flue-cured (line VA-81 and cultivar PD-4), and Connecticut broadleaf (cultivar C9) tobacco (Nicotiana tabacum) resistant (R) or susceptible (S) to the tobacco cyst nematode Globodera tabacum tabacum were crossed. F1 progeny of burley and susceptible broadleaf were selfed and backcrossed to produce additional progeny for evaluation of resistance in greenhouse experiments. Plants without adult female nematodes visible (×10 magnification) on the root surface 6 weeks after inoculation were classified as resistant, whereas those plants in which one or more females were evident were classified as susceptible. Segregation ratios for progeny of resistant and susceptible plants were not different from 3:1 and 1:1 for F2 (F1 × F1) and BC1 (F1 × S) lines, respectively, indicating that resistance in burley to G. t. tabacum is conferred by a single, dominant gene. Segregation ratios for resistance in crosses between nematode-resistant burley and flue-cured tobacco (F1 and F2 progeny) and between burley-flue-cured hybrids and broadleaf BC1 (F1 × S) and BC2 (BC1 × S) progeny were consistent with the assumption that resistance to G. t. tabacum in burley and flue-cured tobacco is conferred by the same or closely linked single, dominant gene(s).     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

Microevolutionary Patterns and Molecular Markers: The Genetics of Geographic Variation in Ascaris suum

Molecular markers have been used only rarely to characterize the population genetic structure of nematodes. Published studies have suggested that different taxa may show distinct genetic architectures. Isoenzyme and RAPD markers have been used to investigate geographic variation of Ascaris suum at the level of infrapopulations (nematodes within individual hosts), within localities, and among geographic regions. Independent estimates of genetic differentiation among population samples based on isoenzyme and RAPD data showed similar patterns and substantial correlation. Heterozygote deficiencies within infrapopulations and large values for inbreeding coefficients among infrapopulations suggested that the composition of these populations was not consistent with a model of random recruitment from a large panmictic pool of life-cycle stages. Both isoenzyme and RAPD markers revealed moderate levels of genetic differentiation among samples representing infrapopulations and localities. Of total gene diversity, 9.4% (isoenzyme) and 9.2% (RAPD) was partitioned among infrapopulations. Geographic localities accounted for 7.8% (isoenzyme) and 6.2% (RAPD) of total diversity. Only infrapopulations from the same farm had low levels of differentiation.     

Molecular Approaches to Taenia asiatica

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.     

Molecular Prevalence of Acarapis Mite Infestations in Honey Bees in Korea

Acarapis mites, including Acarapis woodi, Acarapis externus, and Acarapis dorsalis, are parasites of bees which can cause severe damage to the bee industry by destroying colonies and decreasing honey production. All 3 species are prevalent throughout many countries including UK, USA, Iran, Turkey, China, and Japan. Based on previous reports of Acarapis mites occurring in northeast Asia, including China and Japan, we investigated a survey of Acarapis mite infestations in honey bees in Korean apiaries. A total of 99 colonies of Apis mellifera were sampled from 5 provinces. The head and thorax of 20 bees from each colony were removed for DNA extraction. PCR assays were performed with 3 primer sets, including T, A, and K primers. Results indicated that 42.4% (42/99) of samples were Acarapis-positive by PCR assay which were sequenced to identify species. Each sequence showed 92.6-99.3% homology with reference sequences. Based on the homology, the number of colonies infected with A. dorsalis was 32 which showed the highest infection rate among the 3 species, while the number of colonies infected with A. externus and A. woodi was 9 and 1, respectively. However, none of the Acarapis mites were morphologically detected. This result could be explained that all apiaries in the survey used acaricides against bee mites such as Varroa destructor and Tropilaelaps clareae which also affect against Acarapis mites. Based on this study, it is highly probable that Acarapis mites as well as Varroa and Tropilaelaps could be prevalent in Korean apiaries.     

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.     

Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

Molecular Identification of Diphyllobothrium nihonkaiense from 3 Human Cases in Heilongjiang Province with a Brief Literature Review in China

Human diphyllobothriasis is a widespread fish-borne zoonosis caused by the infection with broad tapeworms belonging to the genus Diphyllobothrium. In mainland China, so far 20 human cases of Diphyllobothrium infections have been reported, and the etiologic species were identified as D. latum and D. nihonkaiense based on morphological characteristics or molecular analysis. In the present study, proglottids of diphyllobothriid tapeworms from 3 human cases that occurred in Heilongjiang Province, China were identified as D. nihonkaiense by sequencing mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 5 (nad5) genes. Two different cox1 gene sequences were obtained. One sequence showed 100% homology with those from humans in Japan. The remaining cox1 gene sequence and 2 different nad5 gene sequences obtained were not described previously, and might reflect endemic genetic characterizations. D. nihonkaiense might also be a major causative species of human diphyllobothriasis in China. Meanwhile, the finding of the first pediatric case of D. nihonkaiense infection in China suggests that infants infected with D. nihonkaiense should not be ignored.     

Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization

Coral disease outbreaks have increased over the last three decades, but their causal agents remain mostly unclear (e.g., bacteria, viruses, fungi, protists). This study details a 14‐month‐long survey of coral colonies in which observations of the development of disease was observed in nearly half of the sampled colonies. A bimonthly qPCR method was used to quantitatively and qualitatively evaluate Symbiodinium assemblages of tagged colonies, and to detect the presence of Vibrio spp. Firstly, our data showed that predisposition to disease development in general, and, more specifically, infection by Vibrio spp. in Acropora cytherea depended on which clades of Symbiodinium were harbored. In both cases, harboring clade D rather than A was beneficial to the coral host. Secondly, the detection of Vibrio spp. in only colonies that developed disease strongly suggests opportunistic traits of the bacteria. Finally, even if sporadic cases of switching and probably shuffling were observed, this long‐term survey does not suggest specific‐clade recruitment in response to stressors. Altogether, our results demonstrate that the fitness of the coral holobiont depends on its initial consortium of Symbiodinium, which is distinct among colonies, rather than a temporary adaptation achieved through acquiring different Symbiodinium clades.     

Morphological and Molecular Characterization of Meloidogyne mayaguensis Isolates from Florida

The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.     

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.     

The Effects of Root-knot Nematode Infection and Mi-mediated Nematode Resistance in Tomato on Plant Fitness

The Mi-1.2 resistance gene in tomato (Solanum lycopersicum) confers resistance against several species of root-knot nematodes (Meloidogyne spp.). This study examined the impact of M. javanica on the reproductive fitness of near-isogenic tomato cultivars with and without Mi-1.2 under field and greenhouse conditions. Surprisingly, neither nematode inoculation or host plant resistance impacted the yield of mature fruits in field microplots (inoculum=8,000 eggs/plant), or fruit or seed production in a follow-up greenhouse bioassay conducted with a higher inoculum level (20,000 eggs/plant). However, under heavy nematode pressure (200,000 eggs/plant), greenhouse-grown plants carrying Mi-1.2 had more than ten-fold greater fruit production than susceptible plants and nearly forty-fold greater estimated lifetime seed production, confirming prior reports of the benefits of Mi-1.2. In all cases Mi-mediated resistance significantly reduced nematode reproduction. These results indicated that tomato can utilize tolerance mechanisms to compensate for moderate levels of nematode infection, but that the Mi-1.2 resistance gene confers a dramatic fitness benefit under heavy nematode pressure. No significant cost of resistance was detected in the absence of nematode infection.     

Inheritance of Resistance to Meloidoygne incognita in Primitive Cotton Accessions from Mexico

Few sources of resistance to root-knot nematodes (Meloidogyne incognita) in upland cotton (Gossypium hirsutum) have been utilized to develop resistant cultivars, making this resistance vulnerable to virulence in the pathogen population. The objectives of this study were to determine the inheritance of resistance in five primitive accessions of G. hirsutum (TX1174, TX1440, TX2076, TX2079, and TX2107) and to determine allelic relations with the genes for resistance in the genotypes Clevewilt-6 (CW) and Wild Mexico Jack Jones (WMJJ). A half-diallel experimental design was used to create 28 populations from crosses among these seven sources of resistance and the susceptible cultivar DeltaPine 90 (DP90). Resistance to M. incognita was measured as eggs per g roots in the parents, F(1) and F(2) generations of each cross. The resistance in CW and WMJJ was inherited as recessive traits, as reported previously for CW, whereas the resistance in the TX accessions was inherited as a dominant trait. Chi square analysis of segregation of resistance in the F(2) was used to estimate the numbers of genes that conditioned resistance. Resistance in CW and WMJJ appeared to be a multigenic trait whereas the resistance in the TX accessions best fit either a one or two gene model. The TX accessions were screened with nine SSR markers linked to resistance loci in other cotton genotypes. The TX accessions lacked the allele amplified by SSR marker CR316 and linked to resistance in CW and other resistant genotypes derived from this source. Four of five TX genotypes lacked the amplification products from the marker BNL1231 that is also associated with the resistant allele on Chromosome 11 in WMJJ, CW, NemX, M120 RNR and Auburn 634 RNR. However, all five TX genotypes produced the same amplification products from three SSR markers linked to the resistant allele on Chromosome 14 in M120 RNR and M240 RNR. The TX accessions have unique resistance genes that are likely to be useful in efforts to develop resistant cotton cultivars with increased durability.     

Molecular identification of a Trichinella isolate from a naturally infected pig in Tibet, China

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.     

The impact of strain diversity and mixed infections on the evolution of resistance to Bacillus thuringiensis

Pesticide mixtures can reduce the rate at which insects evolve pesticide resistance. However, with live biopesticides such as the naturally abundant pathogen Bacillus thuringiensis (Bt), a range of additional biological considerations might affect the evolution of resistance. These can include ecological interactions in mixed infections, the different rates of transmission post-application and the impact of the native biodiversity on the frequency of mixed infections. Using multi-generation selection experiments, we tested how applications of single and mixed strains of Bt from diverse sources (natural isolates and biopesticides) affected the evolution of resistance in the diamondback moth, Plutella xylostella, to a focal strain. There was no significant difference in the rate of evolution of resistance between single and mixed-strain applications although the latter did result in lower insect populations. The relative survivorship of Bt-resistant genotypes was higher in the mixed-strain treatment, in part owing to elevated mortality of susceptible larvae in mixtures. Resistance evolved more quickly with treatments that contained natural isolates, and biological differences in transmission rate may have contributed to this. Our data indicate that the use of mixtures can have unexpected consequences on the fitness of resistant and susceptible insects.     

A Rare Human Case of Dirofilaria repens Infection in the Subcutaneous Posterior Thorax with Molecular Identification

The emergence of Dirofilarial infections in Asia including Vietnam is a clinically significant threat to the community. We here report a rare case of subcutaneous Dirofilaria repens infection on the posterior thoracic wall in a young woman presenting a painful, itchy, and palpable nodule. The adult worm was identified by mitochondrial cox1 and nuclear ITS-2 sequence determination. The diagnosis was additionally confirmed by 16S rRNA sequencing of the endosymbiont Wolbachia pipientis commonly co-existing with D. repens. This is a rare case of subcutaneous human infection on the posterior thoracic region caused by D. repens.     

Breeding Plants for Resistance to Nematodes

Plant breeders and nematologists have developed improved cultivars of important crop species with resistance to plant-parasitic nematodes. The effectiveness of these breeding efforts has depended on the availability of efficient screening procedures, identification of adequate sources of durable resistance, nature of the nematode feeding habit, and knowledge of the inheritance of resistance. These factors determine to a large degree the breeding method and potential success of the research. Systematic searches for nematode resistance have identified resistant germplasm lines within crop species or from related species. When the resistance gene(s) is from related species, incongruity barriers or sterility of the resulting hybrids often must be overcome. In these situations, backcrossing is usually necessary to incorporate the resistance gene(s) and recover the desirable commercial traits of the crop species. If the resistance gene(s) is present within the crop species, the choice of breeding method depends on the inheritance of the resistance, type of screening procedure, and other important breeding objectives for the species. In the future, plant molecular biologists and geneticists will make available novel sources of nematode resistance through incorporation of transgenes from other genera. These efforts will likely require conventional breeding strategies before commercial utilization of an improved resistant cultivar.     

Molecular Detection of Ancylostoma duodenale,Ancylostoma ceylanicum,and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.