Screening for resistance to potato cyst nematodes using bioluminescent photometry

The measurement of the adenosine triphosphate (ATP) content of cysts by bioluminescent photometry has been used as a screen for assessing the resistance of five potato clones to Globodera rostochiensis Rol and G. pallida Pa3. The multiplication rates (P_f/P_i) of the nematodes are based on the ratio of ATP levels for the cyst inoculum and their progeny. These have been compared to the corresponding ratios based on conventional visual counts of eggs and juveniles. The use of recoverable standard volumes of cysts to inoculate pots has allowed an assessment of the unhatched proportion (C_p) of juveniles from the parental cysts by both methods and an estimate of the corrected multiplication rate (P/P^l-Cp)). This is an accurate assessment of resistance for a clone and the use of standard inocula and the ATP method will enable the criterion adopted for establishing resistance to be measured rapidly and precisely.     

Of mudsnails,terrapins, and flukes: Use of trematodes as a field‐based project in parasitology research

We present an exercise for counting trematode cysts on mudsnails that can be implemented as a field‐based project in course work by students or by volunteers. The exercise involves the digenetic trematode Pleurogonius malaclemys, which infects diamondback terrapins (Malaclemys terrapin) as its definitive host, and eastern mudsnails (Tritia obsoleta) as its intermediate host. The trematode forms macroscopic metacercarial cysts on the shells and opercula of the mudsnails, and the life cycle is completed when terrapins ingest the intermediate hosts with cysts. Previous research has suggested that quantifying cysts on mudsnails can be a relatively simple method to estimate terrapin population sizes where terrapins co‐occur with mudsnails. Three non‐professional scientist groups (including students and volunteers) were tested in their estimates of prevalence and intensity data compared to verified cyst counts. Prevalence data were relatively accurate (~76–78%) and reliable (68–89%) across the groups. The intensity of cysts (on shells and opercula of mudsnails) were mostly within 12% of the verified counts (total mean intensity of 1.81–2.97 cysts/parasitized mudsnail) but varied significantly in two cases due to false positives (likely due to sand grains or debris scored as cysts) or negatives (likely due to missed cysts behind the operculum). The exercise can be completed as a lab or series of labs in courses, and data from such projects could be used in terrapin conservation efforts, particularly in helping scientists determine where terrapins occur. We discuss modification of the exercise for other trematode species in regions around the world and hope this expands recognition by the public of the important role of parasites in ecosystems.     

Asymmetric hybridization between Nicotiana tabacum and N. repanda by donor recipient protoplast fusion: transfer of TMV resistance

Summary Genetically asymmetric hybrids were recovered by fusion of Nicotiana tabacum protoplasts with irradiated protoplasts of kanamycin-resistant, nopalineproducing plants of N. repanda. Hybrid calli were selected by culture on media containing kanamycin and were regenerated. These plants were morphologically similar to N. tabacum but produced nopaline, indicating they retained genes from N. repanda. Esterase isozyme profiles also indicated that the plants are somatic hybrids, but are more similar to N. tabacum than N. repanda. Chromosome counts showed most of the hybrids had 55–62 chromosomes, which is consistent with extensive, although incomplete elimination of N. repanda chromosomes. The hybrids were largely male sterile, but about half of them set seed when crossed with N. tabacum. Chromosome numbers of the progeny and the pattern of inheritance of kanamycin resistance indicated the continued elimination of N. repanda genetic material in these backcrosses. The N. repanda parent used in these fusions gave a hypersensitive response to TMV, whereas the N. tabacum parent was TMV sensitive. When inoculated with TMV, plants from two hybrid clones gave a hypersensitive response. Plants from the other clones became systemically infected with the virus.     

A greenhouse technique to screen pigeonpea for resistance to Heterodera cajani*

Heterodera cajani is an important nematode pest of pigeonpea in India and a simple and reliable greenhouse procedure has been developed to screen pigeonpea genotypes for resistance to it. In pot experiments, white cysts of H. cajani were counted on the roots of the susceptible genotype ICPL 87 at 15, 30 and 45 days after seedling emergence in soils infested with different levels of H. cajani. The seedlings were rated for the number of white cysts per root system on a one (highly resistant, no cysts) to nine (highly susceptible, more than 30 cysts) scale. White cysts were not easy to see on wet roots but were clearly visible on slightly dried roots. Cyst counts and ratings were more uniform when roots of 30 day old seedlings were evaluated than when 15 or 45 day old seedlings were examined. Effects of different H. cajani infestation levels on the ratings were not significant although the use of higher inoculum densities (16 to 27 eggs and juveniles/cm³ soil) was effective in reducing variability. This procedure was used to screen 60 pigeonpea genotypes and all of them were rated seven or nine. Ten accessions of Atylosia spp. and Rhynchosia spp. were rated three.     

Preliminary observations of cyst-theca relationships in Spiniferites ramosus and Spiniferites membranaceus (Dinophyceae)

Motile thecal cells derived from the hatching of single cysts identified as Spiniferites membranaceus and S. ramosus have been used to establish cultures. These cultures were examined in order to assess the cyst-theca relationships of these two taxa. The cultures produced two different motile Gonyaulax species belonging to Kofoid's Spinifera group. These cultures were then induced to form a new cyst generation under uniform conditions, and examination of large numbers of the resulting cysts has shown that process development is an extremely variable phenomenon although process morphologies display a continuum within a species. Process length (and to a certain degree, process morphology) requires careful interpretation when used to discriminate Spiniferites taxa, in both modern and ancient environments.     

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.     

Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion

Summary Somatic hybrids were produced between Nicotiana tabacum and N. nesophila, two species incapable of conventional sexual hybridization. Sexual hybrids, though, could be produced between these two species by using ovule culture only when N. nesophila was female. Clones of somatic hybrids were compared with sexual hybrids. Statistically significant variation was observed between clones, but not between sexual hybrids, for pollen viability, flower morphology, leaf morphology, and trichome density. As all clones of somatic hybrids have 96 chromosomes, the variability could not be explained by interclonal variation in chromosome number. Variation between somatic hybrids could be the result of cytoplasmic segregation or recombination, mitotic recombination or small chromosomal rearrangements prior to plant regeneration. Variation between clones could be exploited as these interspecies hybrids are now being used to incorporate disease resistance into cultivated tobacco.     

The relationship between glycoalkaloids and resistance to the white potato cyst nematode, Globodera pallida in potato clones derived from Solanum vernei

No relationship was found between the degree of resistance to Globodera pallida and total glycoalkaloid content of the roots or tubers of a number of potato clones derived from Solanum vernei × S. tuberosum. Nematode infestation of the roots did not lead to increases in the glycoalkaloid content of susceptible or resistant potatoes.     

Construction of Beta procumbens-specific DNA probes and their application for the screening of B. vulgaris x B. procumbens (2n = 19) addition lines

Summary Beta procumbens-specific DNA probes have been constructed by cloning digested total DNA in E. coli and screening the resulting recombinant plasmids in dot blot hybridizations with labelled B. procumbens and B. vulgaris DNA. Four clones (pTS1-4) have been analyzed in detail determining their degree of specificity and DNA sequence. Two clones (pTS1 and pTS2) with the highest degree of B. procumbens specificity were adapted for the squash dot hybridization with the aim of screening large numbers of individual hybrid plants (B. vulgaris x B. procumbens) carrying an alien B. procumbens chromosome (2n = 19). These addition lines carry in some cases B. procumbens resistance genes to the beet cyst nematode (Heterodea schachtii Schm.).     

CHARACTERISTICS OF SEXUAL AND ASEXUAL RESTING CYST (STATOSPORE) FORMATION IN DINOBRYON CYLINDRICUM IMHOF (CHRYSOPHYTA)1

The reproductive significance of siliceous cysts (statospores) produced by the common vernal chrysophyte Dinobryon cylindricum Imhof has been investigated under defined culture conditions. Three types of statospores have thus far been induced in culture: 1) uninucleate, asexual; 2) binucleate, asexual (potentially autogamic); 3) binucleate, sexual (zygotic). The production of each type of cyst responds differently to an array of nutrient deficiencies (P, N, vitamins, micrometals). An individual clone may be capable of participating in the production of all or only a subset of these types of resting cysts. All D. cylindricum statospores are morphologically identical regardless of their reproductive significance. Sexual reproduction leading to zygotic statospore formation is anisogamous, heterothallic, and involves a gametogenic hormone (erogen) that is apparently continuously released from female clones. Only a single bipolar mating group is documented here and clonal compatibility varies considerably within the mating group. The dynamics of encystment for each type of statospore has been determined relative to the growth of the vegetative cell population. Statospores may be produced either during the exponential phase (intrinsic encystment) or stationary phase (extrinsic encystment) of culture growth depending on the clones involved. The effect of both asexual and sexual resting cyst production on the net growth rate and dynamics of natural chrysophyte populations is discussed. Statospores appear to represent a more flexible reproductive strategy than the resistant zygospores produced by the other common groups of planktonic microalgae.     

Aspects of the population dynamics of Neosarmatium meinerti at Mgazana, a warm temperate mangrove swamp in the East Cape, South Africa, investigated using an indirect method

Encysted embryos (cysts) of the brine shrimp, Artemia provide an excellent model system for the study of biochemical adaptation to environmental extremes. Here, we describe an experiment in which cysts of A. franciscana from the San Francisco Bay (SFB), California, U.S.A., were inoculated into experimental ponds in the Mekong Delta region of Vietnam where water temperatures are much higher than the SFB. Cysts produced in each of three successive growing seasons (1996–1998) were collected and examined in the laboratory for resistance to high temperature and relative contents of three stress proteins (Hsp-70, artemin and p26). Thermal adaptation took place rapidly, during the first growing season. The increase in thermal tolerance was reflected in an overall increase in stress protein content, compared to SFB cysts used for the initial inoculation. Also examined were cysts of A. tibetiana collected from a lake on the high plateau of Tibet, PR China, almost 4.5 km above sea level. These cysts were very sensitive to high temperatures, and contained much lower levels of all stress proteins examined, compared to A. franciscana cysts from SFB and Vietnam. Cysts of A. sinica, collected from a hypersaline lake in Inner Mongolia, PR China, were examined in the same fashion and found to be similar to SFB cysts in terms of thermal resistance and stress protein content. The harsh environments in which Artemia are found, and the great diversity of its habitats, world-wide, provide excellent opportunities to relate the ecological setting of an organism to the underlying physiological and biochemical processes enabling its survival.     

Conspecific thistle plant selection by a herbivorous ladybird beetle, Epilachna pustulosa

Host selection by Epilachna pustulosa Kôno (Coleoptera: Coccinellidae) was surveyed in an area of about 130 ares, focusing on the role of the spatial distribution pattern of the host plant, thistle Cirsium kamtschaticum Ledeb. (Asteraceae) and the environmental conditions of habitats where thistle plants were growing. A total of 198 thistle clones were found in the area studied, and approximately 40% showed some degree of E. pustulosa infestation by July. Eggs were only oviposited on thistle clones that were fed on by adults. Adult beetles and egg masses of E. pustulosa showed an aggregated distribution in the earlier season (June) among thistle clones. The distribution of adults became more random (but still aggregated) by the later season (July), along with an increase in the number of infested clones. Multiple logistic regression analyses revealed that clone size and soil moisture were consistently important for the beetle's choice of clones to feed on. The other logistic regression analyses indicated that thistle‐clone size and sunlight condition influenced egg distribution. Thistle clone selection by E. pustulosa changed with season from a rather strict selection in June to a more obscure one in July, expanding the range of thistle clones used as feeding and oviposition substrate.     

Loss of penicillin resistance inParacoccus denitrificans induced by mitomycin C and acridine orange

In an attempt to eliminate the penicillin resistance gene ofP. denitrificans by curing agents, such as acridine orange (AO) and mitomycin C, it was observed that AO treatment caused temporary phenotypic curing where development of sensitivity was a function of concentration of both the curing agent and benzylpenicillin. However, curing with mitomycin produced sensitive clones at a frequency of 6×10⁻³ and two permanently cured clones were isolated. Heavy metal resistance and resistance to other drugs, however, remain unchanged in the mitomycin-cured isolate.     

Fetal bovine serum simultaneously stimulates apoptosis and DNA synthesis in premeiotic stages of spermatogenesis in spiny dogfish (Squalus acanthias) in vitro: modulation by androgen and spermatogenic activity status

Using the simple cystic spermatogenesis in the shark testis as a model, we previously reported the relative resistance of immature spermatogonia (stem cell and early-stage spermatogonia) to apoptosis in the normal testis and after spermatoxicant exposure in vivo. Apoptosis was monitored by fluorescence image analysis of living cysts, using the validated acridine orange (AO) vital staining technique. Findings show that FBS simultaneously stimulates both apoptosis and [³H]thymidine incorporation in immature spermatogonial clones in a concentration-dependent manner in vitro. Furthermore, androgen inhibits apoptosis and increases cyst viability, more so with 10% FBS than with 1% FBS. All the effects were as a function of spermatogenic activity status but were distinct in early-stage spermatogonial cysts isolated from testes awakening from the previous winter spermatogenic arrest period. Results are discussed in the context of the alternating germ–Sertoli cell population kinetics of early-stage spermatogonial cysts in Squalus acanthias’s protracted testicular cycle.     

MORPHOLOGICAL VARIABILITY IN POPULATIONS OF CHRYSOPHYCEAN RESTING CYSTS. I. GENETIC (INTERCLONAL) AND ENCYSTMENT TEMPERATURE EFFECTS ON MORPHOLOGY1

Many chrysophycean species produce resting cysts (statospores) with purportedly species-specific morphology. I investigated variation in the cyst morphology of a single species that may result from genetic differences among the vegetative clones involved and from variation in the temperature of the environment during cyst development. Populations of Dinobryon cylindricum Imhof cysts were produced under defined conditions in vitro and then sampled for morphological analysis based on SEM micrographs. Morphological data is presented and then used in a multivariate discriminant analysis to determine the utility of each morphological character in distinguishing the six populations studied. Results suggest that some features of cyst morphology (i.e. cyst diameter) are invariant among the populations, while other features show distinctive variation. The density of spines covering the cyst body as well as the morphology of those spines appear correlated to the specific clones involved, and thus may represent useful phenotypic genetic markers. The length and definition of both the spines and the cyst collar, on the other hand, are markedly influenced by encystment temperature. The implications of these findings for paleoecological studies is discussed.     

Expression of bovine lactoferrin cDNA confers resistance to Erwinia amylovora in transgenic pear

The siderophore produced by Erwinia amylovora, the causal agent of fire blight of Maloideae, is one of the virulence factors of this bacterium. The production of siderophores enables E. amylovora to overcome the conditions of iron limitation met in plant tissue, and may also protect the bacteria against active oxygen species produced through the Fenton reaction. In this paper, we have examined the ability of an iron chelator protein, encoded by the bovine lactoferrin gene, to reduce fire blight susceptibility in pear (Pyrus communis L.). Transgenic pear clones expressing this gene controlled by the CaMV35S promoter were produced by Agrobacterium tumefaciens mediated transformation. Transformants were analysed by RT-PCR and western blot to determine lactoferrin expression levels. Most transgenic clones demonstrated significant reduction of susceptibility to fire blight in vitro and in the greenhouse when inoculated by E. amylovora. These transgenic clones also showed a significant reduction of symptoms when inoculated with two other pear bacterial pathogens : Pseudomonas syringae pv. syringae and Agrobacterium tumefaciens. Moreover, we have shown that this increase in bacterial resistance was correlated with an increase in root ferric reductase level activity and leaf iron content. Despite negative effects on the growth of a few clones, our results indicate the potential of lactoferrin gene transformation to protect pear from fire blight through increased iron chelation.     

Intraspecific variation of Myzus persicae on sugar beet (Beta vulgaris)

Differences in inherited resistance among seven sugar-beet stocks had similar effects on Myzus persicae clones representing the range of variation in aphid response to resistant and susceptible sugar beet observed in fifty-eight clones collected between 1969 and 1971. Three sugar-beet stocks were consistently resistant. Statistically significant interactions between beet stocks and aphid clones did not indicate the existence of biotypes with specific abilities to overcome resistance. M. persicae clones differed in their vigour of colonizing sugar beet, irrespective of the differences between beet stocks. The readiness of adult aphids to settle determined the size of aphid population produced and included a component related to the response of the aphid clone to sugar beet as a host, and a component related to the resistance ranking of the beet stock. Breeding sugar beet with resistance to aphids will be simplified, as the results indicate that, at present, differences between aphid biotypes need not be considered a problem.     

Identification of AFLP and SSR markers associated with quantitative resistance to Globodera pallida (Stone) in tetraploid potato (Solanum tuberosum subsp. tuberosum) with a view to marker-assisted selection

 Seventy eight clones from the cross between SCRI clone 12601ab1 and cv Stirling were used to explore the possibility of genetical linkage analysis in tetraploid potato (Solanum tuberosum subsp. tuberosum). Clone 12601ab1 had quantitative resistance to Globodera pallida Pa2/3 derived from S. tuberosum subsp. andigena. The strategy adopted involved identifying single- (simplex) and double- (duplex) dose AFLP markers in the parents from segregation ratios that could be unambiguously identified in their offspring, detecting linkage between a marker and a putative quantitative trait locus (QTL) for resistance, and placing the QTL on the linkage map of markers. The numbers of scorable segregating markers were 162 simplex ones present only in 12601ab1, 87 present in Stirling, and 32 present in both; and 72 duplex markers present only in 12601ab1 and 45 present in Stirling. The total map length was 990.9 cM in 12601ab1 and 484.6 cM in Stirling. A QTL with a resistance allele present in double dose (QQqq) in 12601ab1 was inferred from the associations between resistance scores (square root of female counts) and two duplex markers linked in coupling, which, in turn, were linked in coupling to four simplex markers also associated with resistance, but to a lesser degree. The largest marker class difference was the one for the duplex marker P61M34=15. It accounted for 27.8% of the phenotypic variance in resistance scores, or approximately 30% of the genotypic variance. Subsequently, this duplex marker was found to be linked in coupling with a duplex SSR allele Stm3016=a, whose locus was shown to be on chromosome IV in a diploid reference mapping population. The other QTLs for resistance segregating in the progeny were not identified for one or more of the following reasons: the markers did not cover the whole of the genome, there were unfavourable repulsion linkages between the QTLs and markers, or the gene effects were not large enough to be detected in an experiment of the size conducted. It is concluded that prospects appear good for detecting QTLs and using marker-assisted selection in a tetraploid potato breeding programme, provided that, in future, the population size is increased to over 250 and more SSR markers are used to complement the AFLPs; the same is likely to be true for other autotetraploid crops. Received: 16 December 1997 / Accepted: 4 March 1998