Histopathogenesis of Galls Induced by Meloidogyne naasi in Wheat Roots

Histopathogenesis of galls induced by Meloidogyne naasi in wheat roots was studied. Large numbers of larvae penetrated wheat root tips within 24 hr; larvae migrated both inter- and intracellularly, causing cortical hypertrophy. Giant cells were formed in the stele around the head of each nematode within 4 to 5 days. Initial pathological alterations in giant cell formation consisted of hypertrophy of protophloem and protoxylem cells, their nuclei and nucleoli. Giant ceils contained 2 to 8 agglomerated multinucleolate nuclei. Synchronous mitotic divisions were first observed 9 days after inoculation. After 21 days, giant cells became highly vacuolate. Observations 40 days after inoculation revealed a complete degeneration of cell contents in many giant cells but their thick walls remained intact. Abnormal xylem completely surrounded the degenerated or partially degenerated giant cells.     

Autoradiographic detection of the uptake of the label from bacterial3H-DNA in relation to the kinetics of the mitotic cycle in barley embryos

Extirped barley embryos were pre-cultivated in aerated liquid nutrient solution for 24 h and then cultivated for 6 h in nutrient solution containing either³H-DNA fromBacillus subtilis or³H-thymidine. After this treatment the embryos were thoroughly washed and transferred to the fresh nutrient medium. Samples were fixed at different intervals up to 24 h. Feulgen squashes were made and covered with autoradiographic emulsion. Microautodiagrams of different parts of the embryos (root meristem, shoot apex plus meristem of the third leaf, second leaf meristem, coleoptile, scutelum) were observed. Labelling of the nuclei after the application of both³H-DNA and³H-thymidine was found in the proliferating parts of the embryos but no label was found in the scutelum. The labelling index values were almost similar in different embryo organs after the treatment with³H-DNA and³H-thymidine. Labelling index and the fraction of labelled mitoses at different intervals after the application of the labelled substances were almost similar after treatment with³H-DNA and³H-thymidine, except some variations due to irrelevant differences in the kinetics of the mitotic cycle. No disappearance of the activity of³H-DNA was observed at different intervals after removal from the labelled solutions during cultivation for other 24 h in non-labelled nutrient medium either containing DNA fromBacillus subtilis or without it. The embryos which were immersed into 0.2% NaCl solution with either one of the labelled compounds did not show any initiation of the S phase nor uptake of³H-DNA. All these results demonstrate that the label from³H-DNA is localized in those cell nuclei which were in the S phase during treatment but they do not yet distinguish unambiguously between the adsorbtion of polymerous DNA or its degradation and reutilization of low-molecular weight products.     

DNA SYNTHESIS IN THE GLIAL CELLS OF SCRAPIE-AFFECTED MOUSE BRAIN

Radioautographic techniques have been used to study the incorporation of [³H]-thymidine into the DNA of glial cells in normal and scrapie-affected mouse brain. Some differences were seen in the morphological distribution and also in the size distribution of labelled nuclei in sagittal sections of normal and affected brain. Counts of the total number of labelled nuclei per sagittal section showed a progressive decline from 50 to 15 per section in normal brain over the 4-month experimental period. However, in scrapie-affected brain the number increased markedly to 120 per section 5-6 weeks after inoculation and stayed at this level for the remaining 9-10 weeks of the incubation period. The significance of these results in relation to the pathogenesis of scrapie is discussed.     

The Influence of Trichoderma harzianum on the Root-knot Fusarium Wilt Complex in Cotton

Wilt-susceptible cultivar ''Rowden'' cotton was inoculated wilh Meloidogyne incognita (N), Trichoderma harzianum (T), and Fusarium oxysporum f. sp. vasinfectum (F) alone and in all combinations in various time sequences. Plants inoculated with F alone or in combination with T did not develop wilt, Simultaneous inoculation of 7-day-old seedlings with all three organisms (NTF) produced earliest wilt. However, plants receiving nematodes at 7 days and Fusarium and Trichoderma at 2 or 4 weeks later (N-T-F, N-TF) developed the greatest wilt between 49-84 days after initial nematode inoculation. During the same period, Fusarium added 4 weeks after initial nematode inoculation (N-F) and Fusarium added 4 weeks after initial simultaneous inoculation of nematode and Trichoderma (NT-F) produced the least wilt. The addition of Fusarium inhibited nematode reproduction. Simultaneous inoculation with nematodes and Trichoderma (NT-) resulted in the greatest root gall development, whereas nematodes alone produced the greatest number of larvae. In comparison with noninoculated controls (CK), treatments involving all three organisms inhibited plant growth, plants inoculated with the nematode alone (N-) or with nematodes and Trichoderma (NT-) simultaneously had greatest root weight. Any treatment involving the nematode resulted in fewer bolls per plant and greater necrosis on roots than the noninoculated checks.     

Effect of Time,Temperature, and Inoculum Density on Reproduction of Pratylenchus thornei in Carrot Disk Cultures

Reproduction of Pratylenchus thornei on carrot disk cultures at different time periods after inoculation, temperature, and initial inoculum density was studied. At 25 C and with an initial inoculum of 25 females per disk, the final nematode population increased with increasing time after inoculation, although the populations after 25 and 50 days were not different. Nematode numbers increased by 1,255-fold and 3,619-fold at 75 and 100 days, respectively. Over 35 days incubation at 15, 20, 25, and 30 C, the nematode multiplied 1.8, 8.4, 10.5, and 0.4 times, respectively. The optimum temperature for reproduction was between 20 and 25 C, and the nematode life cycle was completed in about 25-35 days. Increasing nematode inoculum (25, 50, 100, 500, 1,000 nematodes per disk) increased the final nematode population but did not increase reproduction rate, the highest being 25.3 at an initial inoculum density of 100 nematodes per disk.     

Post-infection development of Meloidogyne incognita on tomato treated with the endophytes Fusarium oxysporum strain Fo162 and Rhizobium etli strain G12

The endophytic fungus Fusarium oxysporum strain Fo162 and the endophytic bacterium Rhizobium etli strain G12 have been shown to enhance plant resistance toward the root-knot nematode Meloidogyne incognita. The individual inoculation of tomato seedlings with these antagonists lead to significant reductions in the number of juveniles that penetrated the root and ultimately the number of galls and egg-masses produced. The present study determined the influence of Fo162 and G12 root colonization on juvenile development inside the root system over time after a synchronized nematode infection. The results showed that 14 and 21 days after nematode inoculation, the development into the third-stage juvenile as well as into the adult-stage was significantly lower in endophyte-treated plants when compared to the untreated control, respectively. In addition, Fo162 and G12 treatment led to a significant reduction in the number of eggs per female 35 days after nematode inoculation. The results demonstrated that both Fo162 and G12 not only reduce M. incognita root penetration, but also reduce their development and reproduction.     

Dynamics of Belonolaimus longicaudatus Parasitism on a Susceptible St. Augustinegrass Host

St. Augustinegrass (Stenotaphrum secundatum) cv FX-313 was used as a model laboratory host for monitoring population growth of the sting nematode, Belonolaimus longicaudatus, and for quantifying the effects of sting nematode parasitism on host performance in two samples of autoclaved native Margate fine sand with contrasting amounts of organic matter (OM = 7.9% and 3.8%). Following inoculation with 50 Belonolaimus longicaudatus per pot, nematodes peaked at a mean of 2,139 nematodes per pot 84 days after inoculation, remained stable through 168 days at 2,064 nematodes per pot, and declined at 210 days. The relative numbers of juveniles and adults demonstrated senescence after 84 days. Root dry weight of nematode-inoculated plants increased briefly to an apparent equilibrium 84 days after inoculation, whereas root weights of uninoculated controls continued to increase, exceeding those of inoculated plants from 84 to 210 days (P < 0.01). At 210 days, uninoculated plants had 227% the root dry weight of inoculated plants. Transpiration of FX-313 was reduced by nematodes (P < 0.0001) at 84 and 126 days after inoculation; reduction was first observed at 42 days and last observed 168 days after inoculation (P < 0.05). OM content affected all plant performance variables at multiple dates, and generally there were no inoculation x OM content interactions. OM content had no effect on nematode numbers per pot, although there was a slight (P < 0.05) increase in the number of nematodes per gram root dry weight in the low-OM soil compared with the high-OM soil.     

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

[³H] DNA fromEscherichia coli and [³H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [³H] thymidine and [³H] DNA treatment which shows that the activity of [³H] DNA is utilized during the S phase. After application of [³H] thymidine, only cell nuclei in S phase were labelled. After the application of [³H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [³H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [³H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [³H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.     

Effects of Inoculum Level and Time of Microcoleus vaginatus on Control of Meloidogyne incognita on Tomato

Application of Microcoleus vaginatus, a blue-green alga (Cyanobacterium) at different levels along with Meloidogyne incognita, second stage larvae, in the rhizosphere of tomato plants; showed that the plant growth as well as yield of tomato were increased and gall formations and nematode populations decreased with the increase in inoculum level of M. vaginatus. An inoculum level of 20 ml endospores suspension of M. vaginatus (2.4 × 10⁶ endospores per ml) per plant was optimum to reduce nematode attack with a population density of 1000 larvae per kg soil. Plant growth and yield of fruits were greatly suppressed and gall formations on roots, and nematode populations in soil were increased when M. incognita larvae added five days prior to M. vaginatus inoculation. On the other hand, when M. vaginatus inoculated ten days before nematode inoculation, suppressive effect of M. incognta on plants was reduced and their population density as well as gall formations were also decreased significantly. The efficacy of simultaneous inoculation of both nematode and M. vaginatus was lied in between two treatments discussed above.     

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled “Parthenon pillars.” In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.     

Reactions of Grape Rootstocks to Pratylenchus vulnus and Meloidogyne spp.

Five grape rootstocks were inoculated with 0, 100, 1,000, and 10,000 Pratylenchus vulnus. Dogridge and Saltcreek supported low average total numbers of P. vulnus, 136-705/pot, at 12 months after inoculation. Growth of both rootstocks was not affected. Harmony, Couderc 1613, and Ganzin 1 supported high average total numbers, 6-856 times the inoculum levels. Numbers in Harmony continued to increase at all levels but reduced root weight only at the 10,000 level after 12 months. Numbers in Couderc 1613 decreased by 15-30% after 12 months, and root weight was reduced at the 10,000 level. In Ganzin 1, total nematode numbers diminished after 12 months but were still at high levels; growth reduction was proportional to numbers of nematodes added. Meloidogyne incognita, M. javanica, and M. arenaria produced galls and egg masses in Harmony and Couderc 1613 only at 36 C. Galling in Ganzin 1 increased with increasing temperature. Galls in Ganzin 1 at 18 C supported mature females after 90 days. Harmony was resistant to M. incognita in single and concomitant inoculations of P. vulnus and M. incognita. At 250 days after inoculation, total numbers of P. vulnus increased above the inoculum level and the 150-day values; increase was greatest in P. vulnus added singly. Neither nematode species affected growth of Harmony.     

Histopathology of Selected cultivars of Tobacco infected with Meloidogyne species

Rates of nematode penetration and the histopathology of root infections in fluecured tobacco cultivars ''McNair-944,'' ''Speight G-28,'' and ''NC-89'' with either Meloidogyne arenaria, M. incognita, M. hapla, or M. javanica were investigated. Penetration of root tips by juveniles of all species into the M. incognita-resistant NC-89 and G-28 was much less than that on the susceptible McNair-944. Few juveniles of M. incognita were detected in resistant cultivars 7 and 14 days after inoculation. Infection sites exhibited some cavities and extensive necrotic tissue at 14 days; less necrotic tissue and no intact nematodes were observed 35 days after inoculation. Although some females of M. arenaria reached maturity and produced eggs, considerable necrosis was induced in the resistant cultivars. Meloidogyne hapla and M. javanica developed on all cultivars, but there was necrotic tissue at some infection sites in the resistant cultivars. The occurrence of single multistructured nuclei in the syncytia of most M. hapla infections differed from the numerous small nuclei found in syncytia caused by the other three species.     

Development of Meloidogyne chitwoodi on Wheat

Postinfection development of Meloidogyne chitwoodi from second-stage juveniles (J2) to mature females and egg deposition on ''Nugaines'' winter wheat required 105, 51, 36, and 21 days at 10, 15, 20, and 25 C. At 25 C, the J2 induced cavities and hyperplasia in the cortex and apical meristem of root tips with hypertrophy of cortical and apical meristem cell nuclei, 2 and 5 days after inoculation. Giant cells induced by late J2 were observed in the stele 10 days after inoculation. Clusters of egg-laying females were common on wheat root galls 25 days after inoculation. Juveniles penetrated wheat roots at 4 C and above, but not at 2 C, when inoculum was obtained from cultures grown at 20 C, but no penetration occurred at 4 C when inoculum was stored for 12 hours at 4 C before inoculation. In northern Utah, J2 penetrated Nugaines wheat roots in the field in mid-May, about 5 months after seedling emergence. M. chitwoodi eggs were first observed on wheat roots in mid-July when plants were in blossom. Only 40% of overwintered M. chitwoodi eggs hatched at 25 C.     

Effect of Meloidogyne incognita on Plant Nutrient Concentration and Its Influence on the Physiology of Beans

Phaseolus vulgaris plants, 3, 8, 11, and 13 days old, were inoculated with 0, 2,000, 4,000, or 8,000 second-stage Meloidogyne incognita larvae and maintained under controlled conditions. The photosynthetic rate and the shoot and root concentration of K, Ca, Mn, Fe, Cu, and Zn were determined by destructive assay at 1-27-day intervals and by nondestructive assay of leaves, stems, and roots at 27 or 28 days after inoculation. In the destructive assay, the concentration of the elements in the plant tissues did not change until 1 week after inoculation. Thereafter, the trend was mostly decreasing for shoot K and Fe and increasing in the root, whereas Ca had the opposite trend in the shoots. Manganese, Cu, and Fe showed variable trends. Generally, the concentration of K and Mn increased, whereas Ca and Fe decreased, with duration of infection in all treatments. Zinc and Cu decreased in the highest nematode treatments. The overall elemental content generally decreased with level of infection from 1 week after inoculation. Photosynthetic rate based on shoot K concentration significantly decreased with level of infection. In most of the nondestructive assays, the concentrations of shoot K, Zn, and Mn decreased, whereas Ca increased with increasing nematode treatment. One of the first effects of the nematode on host physiology appears to be a change in concentration of nutrient elements in the host plant.     

Relationship of Bursaphelenchus xylophilus Population Density to Mortality of Pinus sylvestris

Seven-month-old Scots pine seedlings were inoculated with water or culture filtrate (controls), with 10,000, or 20,000 (experiment 1), and with 2,500 (experiment 2) Bursaphelenchus xylophilus B.C. isolate nematodes and maintained under defined experimental conditions. Controls did not develop pine wilt disease over a 2-month period. In experiment 1, less than 50% of the inoculum was recovered from the nematode-inoculated seedlings in the first 48 hours, after which the nematode population of both treatments increased exponentially resulting in pine death and approximately equal populations at 216 hours after inoculation. In the second experiment, plant mortality, which was always preceded by 2-3 days of chlorosis and associated stem vascular necrosis, first occurred 14 days after inoculation. The nematode population increased until about day 40 after inoculation and declined thereafter. Nematodes extracted from the roots 2 weeks after inoculation accounted for ca.15% of the total number of nematodes per pine. The study indicates that the rate of nematode reproduction is a factor in pine wilt disease. However, the lack of a linear correlation between the number of nematodes and the timing of pine mortality suggests that the timing of pine death may also depend on the location of nematode damage to the host tissue.     

Hybridizable ribonucleic acid of rat brain

1. Cerebral RNA of adult and newborn rats was labelled in vivo by intracervical injection of [5-³H]uridine or [³²P]phosphate. Hepatic RNA of similar animals was labelled by intraperitoneal administration of [6-¹⁴C]orotic acid. Nuclear and cytoplasmic fractions were isolated and purified by procedures involving extraction with phenol and repeated precipitation with ethanol. 2. The fraction of pulse-labelled RNA from cerebral nuclei that hybridized to homologous DNA exhibited a wide range of turnover values and was heterogeneous in sucrose density gradients. 3. Base composition of the hybridizable RNA was similar to that of the total pulse-labelled material; both were DNA-like. 4. Pulse-labelled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labelled precursor. This finding suggested that cerebral nuclei contained a hybridizable component that was not transferred to cytoplasm. 5. The rates of decay of the hybridizable fractions of cerebral nuclei and cytoplasm were faster in the newborn animal than in the adult. Presumably a larger proportion of labile messenger RNA molecules was present in the immature brain. 6. Cerebral nuclear and cytoplasmic RNA fractions from newborn or adult rats, labelled either in vivo for periods varying from 4min. to 7 days or in vitro by exposure to [³H]-dimethyl sulphate, uniformly hybridized more effectively than the corresponding hepatic preparation. These data suggested that a larger proportion of RNA synthesis was oriented towards messenger RNA formation in brain than in liver.     

The effect of Meloidogyne incognita on the histopathology of Momordica charantia roots

Bitter gourd (Momordica charantia L.) was inoculated with root-knot nematode Meloidogyne incognita to investigate the anatomical abnormalities in the affected roots. Soon after inoculation the second-stage juveniles (J2) entered at or near the root caps and migrated intercellularly towards the zone of vascular differentiation. Discrete giant cells were observed after three days of inoculation. The nematode induced hypertrophy and hyperplasia near the giant cells. After six days, the juveniles moulted to their third stage (J3). At the same, time giant cell size and density of giant cell cytoplasm increased. The continuity of vascular strands remained unaffected. Between 12 and 24 days of inoculation the giant cells enlarged several times and became multinucleate and enclosed dense and granular cytoplasm. The nematodes became almost pyriform 18 days after inoculation. The orientation of vascular strands changed, due to hypertrophy, hyperplasia and enlargement of the nematode. After 30 days of inoculation the nematodes developed into mature females and started egg laying. A large amount of parenchyma transformed into abnormal xylem.     

Annulate lamellae in giant cells surrounding nematode parasites (Thelazia) of the face fly,Musca autumnalis

Annulate lamellae were found in giant cells produced by the face fly, Musca autumnalis, in response to parasitization by the nematode Thelazia sp. These membranous organelles were not found in giant cells 3 or 9 days postinfection, but appeared at 6 days when there was a concommitant increase in rupturing of the nuclear envelope of host nuclei located within the syncytium. Electron micrographs showed direct continuity between annulate lamellae and both smooth and rough endoplasmic reticula. Also, numerous polyribosomes and mitochondria were always associated with this membranous organelle.     

Postembryonic Development of the Redwood Nematode,Rhizonema sequoiae (Nemata: Heteroderidae)

Second-stage larvae of Rhizonema sequoiae Cid del Prado Vera et al. developed into adult females in 6 months or adult males in 3 - 4 months on roots of Sequoia sempervirens maintained in a growth chamber at 16 C with a 12-hour light period. Under these conditions the second-stage larvae increased in diameter, the central cells of the genital primordium increased in size, and their nuclei enlarged. Mesenchymal cells accumulated in the esophageal and tail regions. Second-stage larvae become third-stage males or females 2 months after inoculation of redwood roots. Their sex could be distinguished by the ratio of length to width of the genital primordium, 3.4 for males and 1.6 for females. The stylet in both sexes became slender, the median bulb became robust and almost spherical, and rings of punctation on the cuticle were evident. Fourth-stage females developed in 3 months from the time of inoculation, and fourth-stage males in slightly less time. At this stage the females were more swollen than the males, the rectum was conspicuous, their reproductive system was in the process of elongation, and the annulation of the cuticle was more evident. The ratio of males to females was 2.3. Mature females were completely inside the roots and did not form cysts. The cuticle was entirely annulated, and the first eggs were detected inside the female 4 months after inoculation and started the production of abundant gelatin-like material. The new generation of second-stage larvae hatched inside the female 2 months after she matured, completing the life cycle in 8 months. The redwood nematode also completed its life cycle in 8 months under greenhouse conditions, but the ratio of males to females increased to 7.4. The entire nematode population died out at 25 C after 6 months. In a Marin County, California, forest, where this nematode occurs naturally, the temperature averaged only 9 C over the November to June period of this study, and the redwood nematode reached the fourth stage with a male-to-female ratio of 1.8.     

Water transport properties of cortical cells in roots of nitrogen- and phosphorus-deficient cotton seedlings

Growth-limiting deficiencies of N or P substantially decrease the hydraulic conductance of cotton (Gossypium hirsutum L.) roots. This shift could result from decreased hydraulic conductivity of cells in the radial flow pathway. A pressure microprobe was used to study water relations of cortical cells in roots of cotton seedlings stressed for N or P. During 10 days of seedling growth on a complete nutrient solution, root cell turgor was stable at 0.4 to 0.5 megapascal, the volumetric elastic modulus increased slowly from 6 to 10 megapascals, and the half-time for water exchange increased from 10 to 15 seconds. In seedlings transferred to N-free solution for 10 days, final values for each of those parameters were approximately doubled. Root cell hydraulic conductivity (cell Lp) was 1.4 × 10⁻⁷ meters per second per megapascal at the time of transfer. In the well-nourished controls, cell Lp decreased over 10 days to 38% of the initial value, but in the N-stressed plants it decreased much more sharply, reaching 6% of the initial value after 10 days. Transfer to solutions without P or with an intermediate level of N also decreased cell Lp. The changes in root cell Lp were consistent with nutrient effects on intact-root water relations demonstrated earlier. However, cell Lp was about half that of the intact root, implying that substantial water flow may follow an apoplastic pathway, bypassing the cortical cells from which these values were derived.