Root responses to cereal cyst nematode (Heterodera avenae) in hosts with different resistance genes

The development of cereal cyst nematode (CCN; Heterodera avenae ) induced syncytia in the host roots of infected resistant bread wheat ( Triticum aestivum cv. AUS10894), diploid wheat ( Aegilops tauschii ), barley ( Hordeum vulgare cv. Chebec and cv. Galleon) and in the susceptible wheat cv. Meering and barley cv. Clipper were studied over a period of 13 d. The resistance to CCN in these cereal plants is conferred by the resistance genes Cre1 in the wheat cv. AUS10894, Cre3 in A. tauschii , Ha2 in barley cv. Chebec and Ha4 in barley cv. Galleon. Anatomical observations were made on the development of the syncytia in CCN-infected wheat and barley roots, which carry each of these four sources of resistance genes. Accelerated development of the syncytia in resistant plants, especially in the barley cultivars, was observed. The sites of syncytia development in susceptible wheat and barley were also closely associated with the vascular tissues in the stele, but less so in the resistant plants. The syncytia in the infected susceptible wheat and barley were also metabolically active at day 13. By contrast, the syncytia of resistant wheat plants carrying the Cre1 or Cre3 genes remained extensively vacuolated and less metabolically active. In barley plants with the Ha2 or Ha4 genes, the syncytia appeared non-functional and in early stages of degeneration by day 13 after inoculation.     

Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.     

The invasion of root tips of cereals by the cereal cyst nematode Heterodera avenue

Newly germinated seedlings of susceptible cultivars of oats, wheat, barley and rye were inoculated with second-stage juveniles of Heterodera avenae in pots of sand. Subsequent examination showed oat root tips to be more commonly invaded, and by a greater range of nematode numbers than the other cereals. A comparison of oats and barley showed that lower nematode numbers in barley were not due to a higher emigration from barley; invasion, establishment and emigration by nematodes all being greater in oats. Second-stage juveniles were more likely to migrate prior to establishment in barley than in oats.     

A cereal cyst nematode (Heterodera avenae Woll.) resistance gene transferred from Aegilops triuncialis to hexaploid wheat

 The cereal cyst nematode (Heterodera avenae) is an important root parasite of common wheat. A high level of resistance was transferred to wheat from Aegilops triuncialis (TR lines) using the cross [(T. turgidum×Ae. triuncialis)×T. aestivum]. Low fertility (3–5 viable kernels per plant) was observed during the process but the surviving hybrid plants were highly vigorous. To obtain stable resistant lines further crosses to T. aestivum were performed. The resistance in TR lines seems to be transferred from the C genome of Ae. triuncialis (genomes CCUU). Ae. triuncialis was highly resistant to the two Spanish populations of H. avenae tested, as well as to four French races and two Swedish populations. The histological analysis showed a hypersensitive reaction in the roots of a resistant TR line inoculated with the Ha71 pathotype of H. avenae, whereas well-formed syncytia were observed in the roots of the susceptible control. Resistance to the H. avenae Ha71 pathotype seemed to be inherited as determined by a single dominant factor in the crosses between resistant TR lines and susceptible cultivars. Received: 11 November 1997 / Accepted: 9 December 1997     

Isolation and characterization of microsatellite loci in the sugar beet cyst nematode Heterodera schachtii

We report the isolation of five microsatellites loci from the sugar beet cyst nematode (Heterodera schachtii). Multilocus genotypes were obtained on individual larvae freshly emerged from cysts. Allelic diversity ranged from four to 27 among the five loci. The primers were tested for cross‐species amplification in six other species of phytoparasitic nematodes of the Heterodera genus. Those molecular markers will be used to study the genetic structure of this obligatory parasite and how it is affected by the use of resistant plants.     

Biology of the hop cyst nematode Heterodera humuli

In an intensively sampled English hop garden, cysts of Heterodera humuli were found in every sample taken. Most occurred in the top 15 cm of soil, decreasing in number by half with every 15 cm in depth. Numbers of second-stage juveniles fluctuated during the vegetative period in a pattern which indicated that more than one generation, possibly two to three, were produced. The generation time was about 1.5 months. In pot experiments under controlled conditions (20°C; 16 h light/day) the life cycle lasted 40 days. The optimal temperature was 20°C for the development of H. humuli in the roots of its host Humulus lupulus. Most H. humuli juveniles invaded hop roots at 15°C, but egg hatch was greatest at 20°C. In moist soil, second-stage juveniles survived for at least 54 days and thereafter invaded roots and reproduced.     

大豆胞囊线虫的休眠

以前研究发现,辽宁地区大豆生长期间及收获期土壤中胞囊孵出的二龄幼虫量很少,推测线虫卵的休眠与大豆生长时期或季节相关。为明确该地区大豆胞囊线虫的休眠特点,2002-2003年采用田间随机多点取样、室内分离及模拟自然条件孵化等方法对大豆胞囊线虫的休眠进行深入研究。结果表明:在生长季节,感病品种辽豆10根围土壤中的白色雌虫、卵囊及褐色的胞囊均可孵出二龄幼虫,且孵化持续时间较长,第21d仍有幼虫孵出,白色雌虫及卵囊内的卵孵化率高于褐色胞囊;不同作物对其根围土壤中胞囊内卵的孵化影响不大,寄主作物大豆、非寄主作物玉米根围及休闲地土壤中的胞囊在条件适宜均可孵出二龄幼虫;季节对胞囊内卵的孵化有较大的影响,出苗期孵化率最高,收获期最低,2周时平均1个胞囊孵出幼虫分别为83.8和9.7条;胞囊皮对线虫卵的孵化有显著的影响。表明沈阳地区大豆胞囊线虫在正常和逆境条件下均有部分卵表现休眠。     

Effect of urea and certain NPK fertilizers on the cereal cyst nematode (Heterodera avenae) on wheat

Two outdoor pot experiments were conducted in two consecutive years under outdoor conditions during the wheat growing season in Saudi Arabia to determine the effects of urea and certain compound fertilizers (NPK), compared to the effects of the nematicide fenamiphos on the cereal cyst nematode (CCN), Heterodera avenae, and wheat growth. The results showed that all of the treatments, except the fertilizer diammonium phosphate (DAP), reduced the number of nematode cysts/root system and increased (P ⩽ 0.05) the dry weight of nematode-infected wheat plants. Fenamiphos and urea resulted in the best control, followed by the NPK fertilizers. The combined application of urea and fenamiphos resulted in the most significant effect in decreasing (P ⩽ 0.05) the number of cysts/root system and increasing (P ⩽ 0.05) the growth of nematode-infected wheat plants.     

Effect of some fertilizers on hatching of cereal cysts nematode,Heterodera avenae

Experiments were conducted in laboratory and pot conditions to determine the effects of urea, di-ammonium phosphate (DAP), single super phosphate (SSP), muriate of potash (MOP) and zinc sulphate (ZnSO₄) on hatching of Heterodera avenae. Two concentrations of each fertilizer were tested in lab for which 10 cysts and 5 ml of each concentration were taken in 5 cm diameter Petri plates. Observations were recorded at weekly intervals up to six weeks. Urea, DAP, SSP and MOP inhibited hatching and ZnSO₄ increased it. After six weeks, hatching was least (5.45%) in higher dose of urea and greatest (46.9%) in higher dose of ZnSO₄. In pot experiment, two doses of urea and single dose of SSP, MOP, and ZnSO₄ were applied in H. avenae-infested soil and WH-1105 wheat was sown. Observations on nematodes in roots, soil and remaining cyst contents were recorded 40 days after sowing. Among all the fertilizers, least nematodes in soil and roots were found at higher dose of urea and greatest number in ZnSO₄.     

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.     

Monoclonal antibodies to the soya bean cyst nematode, Heterodera glycines

In order to identify proteinaceous secretions involved in feeding of the soya bean cyst nematode, Heterodera glycines, over 1800 monoclonal antibodies (MAbs) were raised in four experiments using extracts of second stage juveniles and adult females as the immunogens. A rapid, indirect immunofluorescent screening procedure is described that enabled the antigens for 100 MAbs/day to be localised in juveniles. A similar immunofluorescent screening procedure for adult females was less successful due to the masking of the fluorophore by autofluorescence; additional approaches, including enzyme linked immunosorbent assays (ELISA), were necessary to aid in the screening process. The immunosuppressive drug, cyclophosphamide, had an effect on the range of antibodies obtained in two experiments. One was designed to favour antibodies specific to the hatched rather than the unhatched juvenile, whereas the second aimed to select those for the anterior rather than the posterior part of the adult female. A large number of MAbs showed well defined specificities and numbers are given in parentheses for those that recognised the following potential sites of secretion into the plant: the subventral pharyngeal glands (84), the dorsal pharyngeal gland (5), the amphidial pouches (10) and the excretory system (15). These MAbs will aid further work to define the role of nematode secretions in host invasion and the feeding process of this cyst nematode.     

Cloning and characterization of two cellulase genes from Lentinula edodes

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.     

The effects of nematode resistant and susceptible spring oat cultivars and aldicarb on the cereal cyst nematode Heterodera avenae and yields in contrasting soil types

The effects of the cereal cyst-nematode, Heterodera auenae Woll. on resistant and susceptible oat cultivars, with and without aldicarb treatment, were compared on a clay-with-flints soil at Rothamsted and a loamy sand at Woburn. At both sites, when H. auenae was extremely scarce, yields were not further enhanced by aldicarb. At Rothamsted aldicarb increased yields by 48–72% when H. auenae averaged 10 eggs/g soil. At Woburn, aldicarb increased yields of both susceptible and resistant varieties by 80–90% with 20 eggs/g. The resistant varieties conferred yield benefits in the following oat crop equal to the residual effects of aldicarb applied before the previous crop, demonstrating that H. auenae was wholly responsible for the yield losses. Nematode resistant oats suffered as much or more damage from root invasion by H. auenae juveniles as the susceptible varieties but the resulting decrease in nematode numbers led to considerable yield improvements in the following year. At Woburn in 1977, when formalin was an added treatment, fewer females were infected by parasitic fungi and post-crop egg numbers were greater.     

Impact of Cre1, Cre8 and Cre3 genes on cereal cyst nematode resistance in wheat

The cereal cyst nematode (CCN; Heterodera avenae), a root disease of cereal crops, is a major economic constraint in many wheat (Triticum aestivum)-growing areas of the world. The objective of this study was to assess the impact of the Cre1, Cre8 and Cre3 genes on CCN resistance. A population of 92 doubled-haploid (DH) lines derived from a cross between wheat cvs. Frame and Silverstar as well as 1,851 wheat breeding lines were screened for CCN resistance at the Primary Industries Research Victoria (PIRVic). A second population of 9,470 wheat breeding lines was screened at the South Australian Research and Development Institute (SARDI). Cre3 had the largest impact on reducing the number of female cysts, followed by Cre1 and Cre8. There was no significant difference in number of cysts between DH lines with or without the Cre8 marker, suggesting that the marker is not perfectly linked to Cre8. The estimated heritabilities were 0.32 in the DH population, 0.48 in the PIRVic data set and 0.32 in the SARDI data set, which confirm that this is a trait of low heritability. The repeatability of CCN resistance improved with an increase in the number of plants assessed per line—up to ten. However, 85–88% of the improvement was achieved with the assessments of the first five plants.     

The global importance of the cereal cyst nematode (Heterodera spp.) on wheat and international approaches to its control

The Cereal Cyst Nematodes (CCNs) are a group of several closely related species which have been documented to cause economic yield loss on rainfed wheat production systems in several part of the world including North Africa, West Asia, China, India, Australia, America and several countries in Europe. The most commonly reported species is Heterodera avenae, however there are at least two other species H. filipjevi and H. latipons are implicated. It is well appreciated that plants under water and nutrient stress suffer greater yield loss. Control of CCNs requires maintaining nematode populations below economic thresholds. Chemicals are not environmentally sustainable or economic and the major emphasis on control has been with host genetic resistance applied with other integrated pest managent options. Unfortunately due to the number of species and pathotype variation genetic control of Cereal Cyst Nematode with plant resistance is complex. Turkey is one of the top ten wheat producers in the world and has identified these nematode as a major biotic constraint in their rainfed wheat systems. In 2001 a new joint intiative was established between CIMMYT International, the Turkish Ministry of Agriculture and (Ukurova University in Adana to understand i) the distribution of cereal nematodes on wheat; ii) assess the economic importance and improve our understanding of the population dynamics iii) culture, screen and assess known sources of resistance and identify new sources to both groups of nematodes; iv) integrate new sources of resistance into bread wheat cultivars for Turkey and International germplasm using conventional and molecular tools; v) investigate other integrated control options such as rotation and different wheat management strategies and finally vi) capacity build scientists to work in this important area. Some highlights of this work will be presented and the newly formed ICCNI - International Cereal Cyst Nematode Initative introduced.     

Aminopeptidase-like activities in Caenorhabditis elegans and the soybean cyst nematode, Heterodera glycines

Aminopeptidase-like activities in crude whole body extracts of the free-living nematode Caenorhabditis elegans and the plant parasitic soybean cyst nematode Heterodera glycines were examined. General characteristics including pH optima, heat lability, and inactivation of enzyme by organic solvent were the same for the two species. All developmental stages of H. glycines exhibited activity. In older females, activity was present primarily in the eggs. Affinity for the substrate L-alanine-4-nitroanilide was the same regardless of the stage examined, and was similar for the two species (m for C. elegans and m for H. glycines). Nearly all (>95%) of C. elegans aminopeptidase-like activity was present in the soluble fraction of the extract, while H. glycines activity was distributed between the soluble and membrane fractions. Specific activities of the soluble enzymes were highest in C. elegans and H. glycines juveniles. The C. elegans enzyme was susceptible to a number of aminopeptidase inhibitors, particularly to amastatin and leuhistin, each of which inhibited aminopeptidase-like activity more than 90% at 90 microm. In H. glycines, aminopeptidase-like activity was inhibited 39% by amastatin at 900 microm. The apparent molecular weight of the soluble C. elegans enzyme is 70-80 kDa. Some activity in H. glycines is present in the 70-80 kDa range, but most activity (80-90%) is associated with a very high molecular weight (>240 kDa) component.     

Changes in FaRP-like peptide levels during development of eggs from the plant-parasitic cyst nematode, Heterodera glycines

The plant-parasitic cyst nematode Heterodera glycines requires a host plant to complete its life cycle, which involves hatching of infective juveniles that parasitize through root entry. A laboratory population of H. glycines grown on soybean, Glycine max, undergoes a sharp increase in maturity between 5 and 6 weeks in culture, as measured by the proportion of eggs containing well developed pre-hatch juveniles (late development eggs) versus eggs without visible juveniles (early development eggs). The median percent of eggs classified as late development, representing all samples taken from 4 to 7 weeks in culture, was 61%. For all samples taken up to 5 weeks, 80% scored below the median. In samples taken after 5 weeks, 15% scored below the median. This shift in population maturity was accompanied by a significant increase (P < 0.01) in the number of hatched juveniles present in each sample. There was also a significant increase (P < 0.02) in amount of FaRP-like peptide detected by specific ELISA. Total FaRP levels increased from 0.18 +/- 0.07 fMol FLRFamide equivalents per ng protein in early development eggs to 0.40 +/- 0.17 in late development eggs. The level remained high in hatched juveniles. HPLC/ELISA detected as many as nine potential FaRPs in H. glycines, two of which were specifically increased (P < 0.005) in hatched juveniles. The association of FaRPs with maturing eggs and the possible involvement of these neuropeptides with juvenile hatching and motility are discussed.