Coiling is Not Essential to Anhydrobiosis by Aphelenchus avenae on Agar Amended with Sucrose

The roles of preconditioning and coiling upon entrance into anhydrobiosis by Aphelenchus avenae were tested via video-assisted analysis at 25²°C. Fourth-stage juveniles or young adults of A. avenae were individually placed on 5% agar containing 0.8 M sucrose. Nematodes became quiescent within 3 hr, then gradually resumed a low level of activity and assumed a coiled posture. High desiccation survival rate was recorded when nematodes were incubated on agar for more than 6 hr; the survival rates were 0%, 3%, 73%, and 92% for 0, 2, 6, and 12 hr on agar, respectively. All nematodes placed on agar for 24 hr or more revived after rehydration following desiccation. Once nematodes were on agar for a sufficient time, no difference in desiccation survival was observed between nematodes taking a coiled posture and those uncoiled artificially. Based on these results, exposure to osmotic stress for 6 hr can prepare A. aveae physiologically for anhydrobiosis, but coiling does not appear to be a physiological requirement for desiccation in survival.     

Cereal Cyst Nematode (Heterodera avenae) on Oats. II. Early Root Development and Nematode Tolerance

The effect of Heterodera avenae infestation on early seminal and lateral root growth was examined in four oat genotypes differing in tolerance to H. avenae. Recently emerged seminal roots were inoculated with a range of H. avenae larval densities, then transferred a hydroponic system to remove the effect of later nematode penetration on root development. Intolerance to H. avenae was assessed in terms of impairment of seminal root extension resulting in fewer primary lateral roots emerging from the seminal root below the zone of juvenile penetration. Tolerant plants infested with H. avenae had longer lateral root systems than infested intolerant plants. The decline in lateral root growth below the penetration zone was partly offset by increased growth above. This did not contribute to tolerance, however, as there were no differences between cultivars for this feature. Nematodes induced earlier nodal root emergence in all cultivars. Nodal root development was most advanced on the most tolerant cultivar.     

Abscisic Acid and Ethylene Increase in Heterodera avenae-infected Tolerant or Intolerant Oat Cultivars

The relationship between root stunting caused by the cereal cyst nematode and levels of two root growth inhibiting hormones, abscisic acid and ethylene, was investigated in aseptically cultured root segments and in intact roots of two oat cultivars differing in tolerance to the nematode. Cultured root segments of oat cultivars New Zealand Cape (tolerant) and Sual (intolerant) were inoculated with sterilized Heterodera avenae second-stage juveniles. Suppressed growth of root axes and emerged laterals following nematode penetration corresponded to an increase in abscisic acid and ethylene in roots of both intolerant and tolerant cultivars. When the experiment was repeated on intact root systems, nematodes retarded root growth of Sual more than New Zealand Cape despite an increase in ABA and ethylene in both cultivars. Abscisic acid and (or) ethylene may be involved in growth inhibition of H. avenae-infected roots but appear to play no direct role in determining tolerance.     

Infection Behavior and Overwintering Survival of Foliar Nematodes, Aphelenchoides fragariae, on Hosta

We studied the pathogenicity and overwintering survival of the foliar nematode, Aphelenchoides fragariae, infecting Hosta spp. Nematodes applied to either lower or upper sides of noninjured and injured hosta leaves were able to infect and produce typical symptoms on nine cultivars. Leaves of only four cultivars (Borschi, Fragrant Blue, Patomic Pride, and Olive Bailey Langdon) showed no symptoms of nematode infection. The nematodes overwintered as juveniles and adults in soil, dry leaves, and dormant buds, but not in roots. Nematode winter survival was higher in dormant buds and soil from the polyhouse than in an open home garden. Of the nematodes found in the dormant buds, 35% to 79% were located between the first two outside layers of the buds. The nematodes tolerated 8 hr exposure to 40°C and −80°C in leaf tissues. Relative humidity influenced nematode migration from soil to leaves. The presence of nematodes only on the outer surface of foliage (leaves and petioles) confirmed the migration of A. fragariae on the surface of the plants. Of the total number of nematodes found on the foliage, 25% to 46% and 66% to 77% were alive at 90% and 100% relative humidity, respectively, suggesting that high moisture is required for the survival and upward movement of nematodes. We conclude that A. fragariae can overwinter in soil, infected dry leaves, and dormant buds and migrate in films of water on the outer surface of the plant during spring to leaves to initiate infection.     

Are Pathogenesis-Related Proteins Induced by Meloidogne javanica or Heterodera avenae lnvasion?

Changes in root- and leaf-soluble proteins were investigated in tomato after invasion by the root-knot nematode Meloidogyne javanica, or in barley and wheat after invasion by the cereal cyst nematode Heterodera avenae. Infection of susceptible tomato plants by M. javanica did not cause any change in the soluble-protein composition of leaves or roots compared with uninoculated plants at an early infection stage. No pathogenesis-related proteins (chitinase, glucanase, or P-14) were induced in the leaf apoplast. Changes in leaf proteins were not observed after invasion of wheat cultivars by H. avenae, whereas, in barley, a few changes in intercellular leaf proteins were recorded in resistant cultivars. These changes, however, were not the same among different H. avenae-resistant cultivars. Protein changes were found at an early stage of infection in barley and wheat roots infected with H. avenae, but no difference was found between resistant and susceptible cultivars.     

Interactions between Heterodera avenae and Pratylenchus neglectus on Wheat

In a long-term field experiment, differential population densities of Heterodera avenae were produced by frequent cropping with resistant (cv. Panema) or susceptible (cv. Peniarth) oat. The two oat cultivars were equally good hosts of Pratylenchus neglectus in a glass house experiment with field soil. On wheat crops grown after oats in field experiments, P. neglectus population densities in roots were higher in plots where H. avenae had been controlled than in plots with moderate infestations (40 H. avenae eggs/g soil). The field observations indicated that the reduction in population densities of P. neglectus coincided with the development in roots of sedentary stages of the cyst nematode. Evidence for an indirect effect of H. avenae on P. neglectus was found in vitro in a split-root experiment. In the same field, grain yields of two wheat cultivars susceptible or resistant to H. avenae, but both susceptible to P. neglectus, was not reduced by P. neglectus. Alternation of H. avenae resistant and susceptible cultivars is a possible way of exploiting the inverse relationship between these nematodes, whilst controlling cyst nematode -populations in intensive cereal production systems.     

Tolerance of Sargassum thunbergii germlings to thermal, osmotic and desiccation stress

The construction of artificial seaweed beds in the intertidal zone is a challenge due to extreme levels of physical stress. In order to provide a basis for the construction using the dispersal of microscopic juveniles, a three-way factorial experimental design was used to evaluate the tolerance of Sargassum thunbergii germlings shortly released from fertile thalli to temperature, salinity and desiccation in this study. Results revealed that temperature, salinity and desiccation significantly affected the growth and survival of germlings. Germlings showed rapid growth with relative growth rate (RGR, % day⁻¹) over 16% when cultured at 25 °C and full immersion in normal seawater. Although growths of germlings subjected to moderate conditions were significantly inhibited, RGRs over 13% were obtained. The RGRs of germlings below 10% were observed only at 35 °C and 9 h desiccation treatments. In comparison to growth, survival was less affected by physical stress. Germlings showed low mortalities below 10% under appropriate conditions (25 °C and 30 °C combined with full immersion), and below 60% under moderate conditions, by the end of experiment. However, the mortality rates increased to over 90% under extreme conditions (9 h desiccation and 35 °C combined with full immersion in salinity of 12). These results showed that S. thunbergii germlings had high tolerance to physical stresses. In addition to the main effects, both two-way and three-way interactions between temperature, salinity and desiccation were significant. Based on the magnitude of effect, desiccation was the predominant factor affecting both growth and survival. According to the results, construction of artificial tanks in natural habitat to minimize desiccation may be an effective strategy for S. thunbergii restoration using germlings.     

Freezing and Desiccation Tolerance in Entomopathogenic Nematodes: Diversity and Correlation of Traits

The ability of entomopathogenic nematodes to tolerate environmental stress such as desiccating or freezing conditions, can contribute significantly to biocontrol efficacy. Thus, in selecting which nematode to use in a particular biocontrol program, it is important to be able to predict which strain or species to use in target areas where environmental stress is expected. Our objectives were to (i) compare inter- and intraspecific variation in freeze and desiccation tolerance among a broad array of entomopathogenic nematodes, and (ii) determine if freeze and desiccation tolerance are correlated. In laboratory studies we compared nematodes at two levels of relative humidity (RH) (97% and 85%) and exposure periods (24 and 48 h), and nematodes were exposed to freezing temperatures (-2°C) for 6 or 24 h. To assess interspecific variation, we compared ten species including seven that are of current or recent commercial interest: Heterorhabditis bacteriophora (VS), H. floridensis, H. georgiana, (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All), S. feltiae (SN), S. glaseri (VS), S. rarum (17C&E), and S. riobrave (355). To assess intraspecific variation we compared five strains of H. bacteriophora (Baine, Fl1-1, Hb, Oswego, and VS) and four strains of S. carpocapsae (All, Cxrd, DD136, and Sal), and S. riobrave (355, 38b, 7-12, and TP). S. carpocapsae exhibited the highest level of desiccation tolerance among species followed by S. feltiae and S. rarum; the heterorhabditid species exhibited the least desiccation tolerance and S. riobrave and S. glaseri were intermediate. No intraspecific variation was observed in desiccation tolerance; S. carpocapsae strains showed higher tolerance than all H. bacteriophora or S. riobrave strains yet there was no difference detected within species. In interspecies comparisons, poor freeze tolerance was observed in H. indica, and S. glaseri, S. rarum, and S. riobrave whereas H. georgiana and S. feltiae exhibited the highest freeze tolerance, particularly in the 24-h exposure period. Unlike desiccation tolerance, substantial intraspecies variation in freeze tolerance was observed among H. bacteriophora and S. riobrave strains, yet within species variation was not detected among S. carpocapsae strains. Correlation analysis did not detect a relationship between freezing and desiccation tolerance.     

A Realistic Appraisal of Methods to Enhance Desiccation Tolerance of Entomopathogenic Nematodes

Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.     

Comparison of Pratylenchus penetrans Infection and Maladera castanea Feeding on Strawberry Root Rot

The interaction of lesion nematodes, black root rot disease caused by Rhizoctonia fragariae, and root damage caused by feeding of the scarab larva, Maladera castanea, was determined in greenhouse studies. Averaged over all experiments after 12 weeks, root weight was reduced 13% by R. fragariae and 20% by M. castanea. The percentage of the root system affected by root rot was increased by inoculation with either R. fragariae (35% more disease) or P. penetrans (50% more disease) but was unaffected by M. castanea. Rhizoctonia fragariae was isolated from 9.2% of the root segments from plants not inoculated with R. fragariae. The percentage of R. fragariae-infected root segments was increased 3.6-fold by inoculation with R. fragariae on rye seeds. The presence of P. penetrans also increased R. fragariae root infection. The type of injury to root systems was important in determining whether roots were invaded by R. fragariae and increased the severity of black root rot. Pratylenchus penetrans increased R. fragariae infection and the severity of black root rot. Traumatic cutting action by Asiatic garden beetle did not increase root infection or root disease by R. fragariae. Both insects and diseases need to be managed to extend the productive life of perennial strawberry plantings.     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Interaction of Pratylenchus penetrans and Rhizoctonia fragariae in Strawberry Black Root Rot

A split-root technique was used to examine the interaction between Pratylenchus penetrans and the cortical root-rotting pathogen Rhizoctonia fragariae in strawberry black root rot. Plants inoculated with both pathogens on the same half of a split-root crown had greater levels of root rot than plants inoculated separately or with either pathogen alone. Isolation of R. fragariae from field-grown roots differed with root type and time of sampling. Fungal infection of structural roots was low until fruiting, whereas perennial root colonization was high. Isolation of R. fragariae from feeder roots was variable, but was greater from feeder roots on perennial than from structural roots. Isolation of the fungus was greater from structural roots with nematode lesions than from non-symptomatic roots. Rhizoctonia fragariae was a common resident on the sloughed cortex of healthy perennial roots. From this source, the fungus may infect additional roots. The direct effects of lesion nematode feeding and movement are cortical cell damage and death. Indirect effects include discoloration of the endodermis and early polyderm formation. Perhaps weakened or dying cells caused directly or indirectly by P. penetrans are more susceptible to R. fragariae, leading to increased disease.     

Carbamate and Organophosphorus Nematicides: Acetylcholinesterase inhibition and Effects on Dispersal

The sensitivities of acetylcholinesterases (ACHE) from the fungus-feeder Aphelenchus avenae and the plant-parasitic species Helicotylenchus dihystera and Pratylenchus penetrans and the housefly, Musca domestica, were compared using a radiometric assay which utilized H³ acetylcholine as a substrate. Nematode ACHE were generally less sensitive to inhibition by organophosphorns and carbamate pesticides than were ACHE from the housefly. ACHE from the plant-parasitic species and A. avenae were generally similar in sensitivity. In soil, carbamates were more toxic than the organophosphorus pesticides to A. avenae. All pesticides tested affected nematode movement, but fenamiphos was more inhibitory than others. The effects on dispersal of nematodes may be an important mechanism in control by some nematicides.     

Desiccation tolerance,longevity and seed-siring ability of entomophilous pollen from UK native orchid species

Background and Aims

Pollinator-limited seed-set in some terrestrial orchids is compensated for by the presence of long-lived flowers. This study tests the hypothesis that pollen from these insect-pollinated orchids should be desiccation tolerant and relatively long lived using four closely related UK terrestrial species; Anacamptis morio, Dactylorhiza fuchsii, D. maculata and Orchis mascula.

Methods

Pollen from the four species was harvested from inflorescences and germinated in vitro, both immediately and also after drying to simulate interflower transit. Their tolerance to desiccation and short-term survival was additionally assessed after 3 d equilibration at a range of relative humidities (RHs), and related to constructed sorption isotherms (RH vs. moisture content, MC). Ageing of D. fuchsii pollen was further tested over 2 months against temperature and RH, and the resultant survival curves were subjected to probit analysis, and the distribution of pollen death in time (σ) was determined. The viability and siring ability, following artificial pollinations, were determined in D. fuchsii pollen following storage for 6 years at –20 °C.

Key Results

The pollen from all four species exhibited systematic increases in germinability and desiccation tolerance as anthesis approached, and pollen from open flowers generally retained high germinability. Short-term storage revealed sensitivity to low RH, whilst optimum survival occurred at comparable RHs in all species. Similarly, estimated pollen life spans (σ) at differing temperatures were longest under the dry conditions. Despite a reduction in germination and seeds per capsule, long-term storage of D. fuchsii pollen did not impact on subsequent seed germination in vitro.

Conclusions

Substantial pollen desiccation tolerance and life span of the four entomophilous orchids reflects a resilient survival strategy in response to unpredictable pollinator visitation, and presents an alternative approach to germplasm conservation.     

Dehydrogenases,Acid and Alkaline Phosphatases,and Esterases for Chemotaxonomy of Selected Meloidogyne,Ditylenchus, Heterodera and Aphelenchus spp.

Various taxonomically useful profiles of four dehydrogenases (lactate, malate, glucose-6-phosphate, and a-glycerophosphate) and three hydrolases (acid and alkaline phosphatase and esterase) were detected in whole nematode homogenates of Meloidogyne javanica, M. hapla, M. incognita, M. arenaria, Ditylenchus dipsaci, D. triformis, Heterodera glycines, and Aphelenchus avenae. The enzyme profiles were stable in populations cultured on several different hosts. A tentative enzymically-determined phylogeny of Meloidogyne is given.     

Anhydrobiosis in Nematodes II: Carbohydrate and Lipid Analysis in Undesiccated and Desiccate Nematodes

Glycogen, trehalose, glucose, and total lipid contents of six nematode species were studied. Anhydrobiotic Anguina tritici and Ditylencbus dipsaci stored trehalose in preference to glycogen and only small amounts of glucose were detected. Glycogen content was also reduced in anhydrobiotic Aphelenchus avenae. Conversely, Panagrellus redivivus and Turbatrix aceti contained large amounts of glycogen, appreciable amounts of glucose, and minimal amounts of trehalose. Ditylenchus myceliophagous "curds" contained low amounts of glycogen and very little trehalose; total lipid was 60% of that in fresh samples. The lipid contents of fresh samples of P. redivivus, T. aceti, and A. avenae were high (23.1, 21.9, and 36.7% dry weight, respectively), but in anhydrobiotic A. avenae larvae the level was reduced by over 60%. In contrast, lipid levels remained high in anhydrobiotic A. tritici and D. dipsaci larvae (40.6 and 38.3%, respectively). Analysis of lipid composition in anhydrobiotic A. tritici and A. avenae did not indicate any specific metabolic adaptations to desiccation survival.     

Biological Control of Soil Pests by Mixed Application of Entomopathogenic and Fungivorous Nematodes

In greenhouse experiments, massive application of the fungivorous nematode, Aphelenchus avenae, in summer at 26-33 C (1 x l0⁵ nematodes/500 cm³ autoclaved soil) or in autumn at 18-23 C (5 x 10⁴ nematodes/500 cm³ autoclaved soil) suppressed pre-emergence damping-off of cucumber seedlings due to Rhizoctonia solani AG-4 by 67% or 87%, respectively. Application of 2 x l0⁵ A. avenae to sterilized soil infested with R. solani caused leafminer-like symptom on the cotyledons, which did not occur in mixed inoculations with the entomopathogenic nematode, Steinernema carpocapsae. When 1 x 10⁶ A. avenae were applied 3 days before inoculation with 100 Meloidogyne incognita juveniles, gall numbers on tomato roots were reduced to 50% of controls. Gall numbers also were suppressed by S. carpocapsae (str. All). Reduction in gall numbers was no greater with mixed application of A. avenae and S. carpocapsae than with application of single species, even though twice the number of nematodes were added in the former case. These nematodes were positively attracted to tomato root tips. Aphelenchus avenae suppressed infection of the turnip moth, Agrotis segetum, but not the common cutworm, Spodoptera litura, by S. carpocapsae.     

Partial Resistance to Heterodera avenae in Wheat Lines with the 6M v Chromosome from Aegilops ventricosa

Lines of wheat with the 6M^v chromosome from Aegilops ventricosa display partial resistance to both pathotypes Hal2 and Ha41 of Heterodera avenae. With either pathotype, the effect of this alien chromosome on cyst production, size, and fecundity was expressed in resistance tests. Partial resistance of five 6M^v(6D) substitution lines varied according to the intrinsic cyst-forming capacity of the nematode pathotypes and the recipient germplasms. Such partial resistance can be utilized in wheat breeding lines for integrated management of the cereal cyst nematode.     

Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.)

Enhancement of callus induction and its regeneration efficiency through in vitro techniques has been optimized for 2 abiotic stresses (salt and air desiccation) using 3 rice genotypes viz. BR10, BRRI dhan32 and BRRI dhan47. The highest frequency of callus induction was obtained for BRRI dhan32 (64.44%) in MS medium supplemented with 2, 4-D (2.5 mgL⁻¹) and Kin (1.0 mgL⁻¹). Different concentrations of NaCl (2.9, 5.9, 8.8 and 11.7 gL⁻¹) were used and its effect was recorded on the basis of viability of calli (VC), relative growth rate (RGR), tolerance index (TI) and relative water content (RWC). It was observed that in all cases BRRI dhan47 showed highest performance on tolerance to VC (45.33%), RGR (1.03%), TI (0.20%) and RWC (10.23%) with 11.7 gL⁻¹ NaCl. Plant regeneration capability was recorded after partial air desiccation pretreatment to calli for 15, 30, 45 and 60 h. In this case BRRI dhan32 gave maximum number of regeneration (76.19%) when 4 weeks old calli were desiccated for 45 h. It was observed that air desiccation was 2-3 folds more effective for enhancing green plantlet regeneration compared to controls. Furthermore, desiccated calli also showed the better capability to survive in NaCl induced abiotic stress; and gave 1.9 fold (88.80%) increased regeneration in 11.7 gL⁻¹ salt level for BRRI dhan47. Analysis of variance (ANOVA) showed that the genotypes, air desiccation and NaCl had significant effect on plant regeneration at P < 0.01.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.