Effects of Temperature on the Fine-Structural Responses in the Hypocotyl Region of Alfalfa Lines to Ditylenchus dipsaci

Fine-structural analyses were made of the response of host tissue, Medicago sativa L. ''Ranger'' and ''Lahontan'', to infection by the stem nematode, Ditylenchus dipsaci (Kühn) Filipjev. Seedlings were grown at 15 and 25 C, and hypocotyl regions were sampled 1,3, or 7 days after inoculation. Electron micrographs of infected alfalfa tissue indicated that the same types of damage were inflicted on Lahontan (stem-nematode-tolerant)and Ranger (stem-nematode-susceptible). Only the infection rate and degree of damage differed between lines and temperatures, with the greater injury occurring at the higher temperatures. After 3 and 7 days of infection, the symptoms observed were: swollen and broken endoplasmic reticulum (ER), distended and broken chloroplasts, loss of nuclear material, and bulging and rupturing of nuclear envelopes. Cells with infected cytoplasm contained more ER, ribosomes, vesicles, and Golgi apparatuses, suggesting increased metabolic activities. Lobing nuclei were observed in all samples. Lipid contents varied with temperature in I-day-old seedlings. At 15 and 25 C, electron-dense substrances were commonly found along the tonoplast, and on the cell wall. Also, some cells with enlarged ER were noted in the noninfected controls at these temperatures.     

Influence of Plant-Parasitic Nematodes on Longleaf Pine Seedlings

Seedlings of longleaf pine (Pinus palustris) were grown in 20-cm pots for 5 to 7 months in the greenhouse following inoculation with a high or low level of one of seven species of plant-parasitic nematodes. Belonolaimus longicaudatus and Helicotylenchus dihystera had no effect on seedling growth. High inoculum densities of Hoplolaimus galeatus and Tylenchorhynchus claytoni caused a significant reduction of fresh weight of seedling roots. Root and top weights of seedlings grown in soil infested with Meloidodera floridensis or Pratylenchus brachyurus were significantly less than those of seedlings in noninfested soil. Root growth of seedlings was stimulated by the higher inoculum density of Scutellonema brachyurum.     

Changes in Alfalfa Stem Conductance Induced by Corynebacterium insidiosum Toxin

A toxin involved in bacterial wilt of alfalfa (Medicago sativa L.) has been isolated from cultures of the pathogen, Corynebacterium insidiosum, as well as from diseased plants (S. M. Ries and G. A. Strobel. 1972. Physiological Plant Pathology 2: 133-142). The influence of this toxin, a glycopeptide with a molecular weight of 5 × 10⁶, on the water relations of alfalfa was examined. It was found that very small amounts of the toxin (2 μg) significantly reduced stem conductance through 15-cm long stems. This decrease in stem conductance caused by the toxin best explains the rapid decrease in transpiration and stomatal conductance and the resultant wilting after alfalfa cuttings have been in 200 μg ml⁻¹ toxin for 2 hours. Membrane damage resulting in water leakage was ruled out as a factor in the wilting during the 2-hour period. It is postulated that the toxin acts by interfering with water movement through pit membranes.     

Morphological and Histochemical Changes Occurring during the Life-span of Root-tip Galls on Lolium perenne Induced by Longidorus elongatus

The RNA and protein content of perennial ryegrass root-tip galls induced by Longidorus elongatus were measured from transverse sections and the morphology described. Galls progressed through five distinct stages and were viable for only 10-12 days at 18 C, after which they collapsed and became necrotic. In the initial stage hypertrophy occurred and cells contained enlarged nuclei and nucleoli, a greater proportion of cytoplasm, and increased concentrations of protein. This was followed by hyperplasia; cells divided to give two or four daughter cells, accompanied by a proportionate reduction in volumes of cytoplasm, nuclei, and nucleoli and reduced concentrations of RNA and protein. The third stage was secondary hypertrophy with enlarged, amoeboid nuclei and nucleoli and a significant increase in concentration of RNA and protein. In the final two stages, as feeding by L. elongatus progressively removed cell contents, most cells were devoid of inclusions and galls collapsed and were invaded by soil bacteria. This ordered development and exploitation of galls suggests that L. elongatus may have two phases in its feeding.     

Interactions Among Selected Endoparasitic Nematodes and Three Pseudomonads on Alfalfa

Meloidogyne hapla, Pratylenchus penetrans, and Helicotylenchus dihystera, reduced the growth of ''Saranac AR alfalfa seedlings when applied at concentrations of 50 nematodes per plant. All except P. penetrans reduced seedling growth when applied at 25 per seedling. M. hapla reduced growth when applied at 12 per seedling. Nematodes interacted with three pseudomonads to produce greater growth reductions than were obtained with single pathogens, suggesting synergistic relationships. Ditylenchus dipsaci, applied at 25 or 50 nematodes per seedling, reduced plant weight compared with weights of control plants, but did not interact with test bacteria. All of the nematodes except D. dipsaci produced root wounds which were invaded by bacteria.     

Sample Optimization for Five Plant-Parasitic Nematodes in an Alfalfa Field

A data base representing nematode counts and soil weight from 1,936 individual soil cores taken from a 7-ha alfalfa field was used to investigate sample optimization for five plant-parasitic nematodes: Meloidogyne arenaria, Pratylenchus minyus, Merlinius brevidens, Helicotylenchus digonicus, and Paratrichodorus minor. Sample plans were evaluated by the accuracy and reliability of their estimation of the population and by the cost of collecting, processing, and counting the samples. Interactive FORTRAN programs were constructed to simulate four collecting patterns: random; division of the field into square sub-units (cells); and division of the field into rectangular sub-traits (strips) running in two directions. Depending on the pattern, sample numbers varied from 1 to 25 with each sample representing from 1 to 50 cores. Each pattern, sample, and core combination was replicated 50 times. Strip stratification north/south was the most optimal sampling pattern in this field because it isolated a streak of fine-textured soil. The mathematical optimmn was not found because of data range limitations. When practical economic time constraints (5 hr to collect, process, and count nematode samples) are placed on the optimization process, all species estimates deviate no more than 25 % from the true mean. If accuracy constraints are placed on the process (no more than 15% deviation from true field mean), all species except Merlinius required less than 5 hr to complete the sample process.     

Interrelationships between Ethylene Production,Gall Formation,and Root-knot Nematode Development in Tomato Plants Infected with Meloidogyne javanica

Ethylene production was determined in excised tomato (Lycopersicon esculentum) root cultures of Meloidogyne javanica susceptible and resistant cultivars infected with M. javanica. Uninfected cultivars produced very low amounts of ethylene. Relatively high amounts of ethylene were produced by the infected susceptible cultivars. Peak production of 1.6 n moles * g root⁻¹ * h¹⁻ occurred between 9 and 16 days after inoculation (DAI). The period of high ethylene production coincided with that of rapid increase in gall weight. Low amounts of ethylene were also released by the infected resistant cultivar between 9 and 12 DAI, which follows the hypersensitivity reaction. Ethylene production in infected intact plants during the period of rapid gall growth was twice as much as in uninfected plants during the same time. Exposing excised root cultures to 0.5 or l0 ppm ethylene accelerated the rate of increase in gall weight of M. javanica infected roots. In contrast, overall root growth was inhibited by these treatments, compared to infected roots which were not exposed to ethylene.     

Interactions Between Nematodes and Other Factors on Plants

The distinction between qualitative and quantitative interactions is stressed as it helps to elucidate differences between two current definitions of the term synergism and how multifactorial experiments should be designed, analysed statistically, and their results interpreted. Factorial design and analyses are strongly advocated so that interactions can be detected. It is suggested that interactions involving nematodes are common in nature and should be included, where appropriate, in hypotheses. Methods for testing the hypothesis that environment influences tolerance are suggested.     

Ultrastructure of Esophageal Gland Secretory Granules in Juveniles of Heterodera glycines

Ultrastructural observations of the feeding sites of soybean cyst nematode juveniles 3 days after inoculation of soybean roots revealed the presence of feeding tubes in the host cell syncytium. Feeding tubes, which were extruded from the stylet tips, were formed by products of secretory granules that originated in the dorsal esophageal gland and accumulated in the ampulla of the gland extension. Granules traversing the space between the gland cell and the ampulla were regulated in their movement by two sets of sphincter-like muscles located anterior and posterior to the metacorpus pump chamber. Sections through the sphincter muscles revealed obliquely arranged fibers, which in a contracted mode caused microtubules in the gland extension to be tightly packed and devoid of granules.     

Changes in Saccharide Metabolism Induced by Infection of Camellia Sinensis by Exobasidium Vexans

Changes in saccharide contents of tea leaves during infection with blister blight fungus Exobasidium vexans Masse was studied. Saccharose and glucose contents decreased in the blistered portions when compared to the normal regions until sporulation and remained constant during the entire period of sporulation. Fructose content increased abruptly during the initiation of sporulation and remained constant up to the end of sporulation in both blistered and non-blistered regions. Starch content continuously decreased in the blistered region. Peroxidase activity was highly enhanced during the final stages of leaf senescence. The activity of acid invertase was inversely proportional to the starch content and closely related to the changes in the saccharose and glucose contents. Protein and chlorophyll contents gradually decreased in the blistered regions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in the Ultrastructure of Adrenocortical Cells Induced by Cholinergic Agonists

The role of cholinergic neurotransmitters in the changes in ultrastructure of adrenocortical cells remains to a great extent unexplored. We studied the influence of acetylcholine (ACh) and pilocarpine, agonists of muscarinic ACh (mACh) receptors, on the ultrastructure of adrenocortical secretory cells derived from the rat adrenal cortex. Both agonists were found to stimulate fragmentation of the mitochondria and lipid droplets and reconstruction of the peroxisomal compartment. At the same time, assembling of the smooth endoplasmic reticulum (sER) membranes was observed after ACh application, while pilocarpine action was not always prominent. Thus, we conclude that activation of mACh receptors by its agonists influence the ultrastructure of the adrenocortical cells, and the respective ACh-induced effects are more intensive.     

低温下的绿豆下胚轴细胞超微结构变化

采用透射电子显微镜观察经10℃低温处理2d的绿豆下胚轴细胞超微结构变化的结果表明,细胞受到不同程度的伤害,其中大部分细胞的损伤是可逆的而非致死性的,如内质网膜模糊和呈颗粒状;线粒体呈现不同程度的膨胀,其数量增加,且聚集分布在内质网和高尔基体附近,核糖体聚集成为多聚核糖体等。也有小部分细胞受到的伤害是致死性的,如细胞质电子密度显著升高,质体中淀粉粒过度积累,内质网和高尔基体膨胀和解体,小液泡频繁吞噬细胞质和细胞器,细胞自溶死亡等。     

Generation of Transgenic Rats through Induced Pluripotent Stem Cells

The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.     

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.     

Similar Metabolic Changes Induced by HIPVs Exposure as Herbivore in Ammopiptanthus mongolicus

Herbivore-induced plant volatiles (HIPVs) are important compounds to prim neighboring undamaged plants; however, the mechanism for this priming process remains unclear. To reveal metabolic changes in plants exposed to HIPVs, metabolism of leaves and roots of Ammopiptanthus mongolicus seedlings exposed to HIPVs released from conspecific plants infested with larvae of Orgyia ericae were analyzed together with control and infested seedlings using nuclear magnetic resonance (NMR)-based metabolic technology and multi variate data analysis. Results presented showed that HIPVs exposure led to similar but specific metabolic changes compared with those induced by infestation in both leaves and roots. Furthermore, both HIPVs exposure and herbivore attack resulted in metabolic changes involving a series of primary and secondary metabolites in both leaves and roots. Taken together, these results suggested that priming of yet-damaged plants may be achieved by reconfiguring metabolic pathways in leaves and roots to make similar concentrations for all metabolites as those in seedlings infested. Therefore, we propose that improved readiness of defense induction of primed plants toward subsequent herbivore attack may be based on the similar metabolic profiling induced by HIPVs exposure as those caused by herbivore.     

Stem Nematode-Fusarium Wilt Complex in Alfalfa as Related to Irrigation Management at Harvest Time

A high moisture level in the top 10 cm of soil at time of cutting of alfalfa increased the incidence of plant mortality and Fusarium wilt in soil infested with Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis in greenhouse and field microplot studies. Ranger alfalfa, susceptible to both D. dipsaci and F. oxysporum f. sp. medicaginis, was less persistent than Moapa 69 (nematode susceptible and Fusarium wilt resistant) and Lahontan alfalfa (nematode resistant with low Fusarium wilt resistance). In the greenhouse, the persistence of Ranger, Moapa 69, and Lahontan alfalfa plants was 46%, 64%, and 67% respectively, in nematode + fungus infested soil at high soil moisture at time of cutting. This compared to 74%, 84%, and 73% persistence of Ranger, Moapa 69, and Lahontan, respectively, at low soil moisture at time of cutting. Shoot weights as a percentage of uninoculated controls at the high soil moisture level were 38%, 40%, and 71% for Ranger, Moapa 69, and Lahontan, respectively. Low soil moisture at time of cutting negated the effect D. dipsaci on plant persistence and growth of subsequent cuttings, and reduced Fusarium wilt of plants in the nematode-fungus treatment; shoot weights were 75%, 90%, and 74% of uninoculated controls for Ranger, Moapa 69, and Lahontan. Similar results were obtained in the field microplot study, and stand persistence and shoot weights were less in nematode + fungus-infested soil at the high soil-moisture level (early irrigation) than at the low soil-moisture level (late irrigation).