Independent resistant reactions expressed in root and tuber of potato breeding lines with introgressed resistance to Meloidogyne chitwoodi.

Resistance to Meloidogyne chitwoodi was introgressed from Solanum bulbocastanum into the cultivated gene pool of potato. A single dominant gene is responsible for resistance to race 1 reproduction on the root system. An additional form of resistance was discovered in certain advanced backcross clones. A BC(5) clone, PA99N82-4, resisted invasion of tubers by available nematode juveniles whether supplied by weeds or challenged by several root resistance-breaking pathotypes. This tuber resistance is inherited as a single dominant gene and is linked to R(Mc1(blb)). Because this gene has been mapped to chromosome 11, tuber resistance genetic factors are inferred to be on the same chromosome in coupling phase. Among 153 progeny derived from crosses with PA99N82-4, 42 recombinants, comprising both resistant root/susceptible tuber and susceptible root/resistant tubers, were found while other progeny were doubly resistant (like PA99N82-4) or doubly susceptible. Therefore, the existence of two linked genetic factors controlling independently expressed traits is confirmed. The combination of the two phenotypes is likely to be a sufficient level of resistance to avoid tuber damage from circumstances that provide exogenous juveniles proximal to the tubers in the soil. These factors are weed hosts of M. chitwoodi host races and pathotypes of M. chitwoodi that overcome R(Mc1(blb)). Under field conditions, where a resistance-breaking pathotype of M. chitwoodi was present, tuber-resistant PA99N82-4 breeding line produced tubers which were commercially acceptable and not culled. A related breeding line, root resistant but tuber susceptible, and Russet Burbank were severely tuber damaged and commercially unacceptable.

Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens.

The stubby root nematode, Paratrichodorus allius, is important to the potato industry in the Pacific Northwest of USA, because it vectors Tobacco rattle virus (TRV), the causal agent of corky ringspot disease. The current method for determining if nematodes are viruliferous for TRV takes several weeks, requiring a glasshouse bioassay followed by a serological test. To overcome this drawback, a rapid and affordable molecular test was developed using reverse transcription polymerase chain reaction (RT-PCR) to identify viruliferous P. allius nematodes within 48 hours. Primers from the 16 kDa gene of TRV were used to detect TRV in both greenhouse-reared and field collected P. allius. TRV RNA can be detected consistently in nucleic acids equivalent to one quarter of a viruliferous adult nematode reared in the greenhouse. In order to reduce the time and expense of processing individual nematodes from field samples, viral RNA was consistently and affordably detected in extracts from 5 field-collected adult P. allius.

Pratylenchus neglectus, P. thornei, and Paratylenchus hamatus Nematodes Causing Yield Reduction to Dryland Peas and Lentils in Idaho.

In June 2006, stunted and chlorotic plants were observed in large patches in two 40.5-ha fields of dryland peas (Pisum sativum) in Latah County, Idaho, which resulted in 90 and 75% crop loss. In the same region, a 121.4-ha field of dryland lentils (Lens culinaris) also had plants showing poor growth, wilting, and yellowing in large patches, which resulted in 40% crop loss. Two species of lesion nematodes (Pratylenchus neglectus and P. thornei) and one species of pin nematode (Paratylenchus hamatus) were extracted from rhizosphere soil and the roots of symptomatic plants from these fields. In a subsequent survey of seven dryland pea fields, under cv. Columbian, in Latah and Nez Perce counties and one dryland pea field, under cv. Small Sieve, in Latah County, plant samples had means of 551 and 2,178 mixed species of lesion nematodes per gram of dry root, respectively. Plant samples from 12 lentil fields in Latah County, six planted with cv. Red Chief and six with cv. Pardina, had means of 279 and 987 mixed species of lesion nematodes per gram of dry root, respectively. Soil samples from the same fields had a mean of 628 and 671 pin nematodes, Paratylenchus hamatus, per 250 cm3 soil for Red Chief and Pardina, respectively. Lentils cv. Pardina and peas cv. Columbian were planted separately in six pots, five seeds per pot containing 250 g of infested soil brought from the field to the greenhouse. Fumigated sandy loam soil was used as control. These assays were repeated three times. In addition, peas and lentils were planted to pots infested singly with each of the three nematode species. For this assay, nematodes were extracted from field soil, surface sterilized, and used to infest 250 g of fumigated sandy loam soil at two nematodes per gram of soil. Six plants per nematode species and an uninoculated control were used in the greenhouse assays, which were repeated three times. Nematodes in all of the assays reduced plant growth in comparison with controls; an average of 50 to 70% reduction in plant height was noted. The lesion nematode populations increased in all pots. The greenhouse assays verified the negative impact of these nematodes on growth of dryland peas cvs. Columbian and Small Sieve and lentils cvs. Red Chief and Pardina. P. neglectus, P. thornei, and Paratylenchus spp. previously have been reported from the semi-arid Pacific Northwest (1). However, to our knowledge, this is the first report attributing plant growth and yield reduction of certain cultivars of lentils and peas to these two species of lesion nematodes and pin nematodes, identified to species level as Paratylenchus hamatus. Reference: (1) R. W. Smiley et al. J. Nematol. 36:54, 2004.

Establishing a corky ringspot disease plot for research purposes.

A method to establish two experimental corky ringspot disease (CRS) plots that had no prior CRS history is described. CRS is a serious disease of potato in the Pacific Northwest caused by tobacco rattle virus (TRV) and transmitted primarily by Paratrichodorus allius. 'Samsun NN' tobacco seedlings were inoculated with viruliferous P. allius in the greenhouse before they were transplanted into the field soil at the rate of 3,000 plus seedlings/ha. Care was taken to keep soil around plants in the greenhouse and transplants in the field moist to avoid vector mortality. The vector population in the soil of one of the fields was monitored by extraction, examination under microscope and bioassay on tobacco seedlings to ascertain that they were virus carriers. Presence of virus in tobacco bioassay plants was determined by visual symptoms on tobacco leaves and by testing leaves and roots using ELISA. Although TRV transmission was rapid, there was loss of infectivity in the first winter which necessitated a re-inoculation. After two years of planting infected tobacco seedlings, 100% of soil samples collected from this field contained viruliferous P. allius. In the second field, all five commercial potato cultivars, known to be susceptible, expressed symptoms of CRS disease indicating that the procedure was successful.

A New Pathotype of Meloidogyne chitwoodi Race 1 from Washington State.

Meloidogyne chitwoodi Golden et al. is a serious pest of potato (Solanum tuberosum L.), and is widespread in the Pacific Northwest United States. M. chitwoodi is currently reported to consist of two host races and one pathotype (2,3) that are not distinguished morphologically. Host race 1 reproduces on Chantenay carrot but not on Thor alfalfa and host race 2 reproduces on alfalfa but not on carrot. Both races fail to reproduce on roots of S. bulbocastanum, a wild potato species used as a source of resistance in our breeding program (1). The resistance to race 1 in S. bulbocastanum is attributed to Rmc1(blb) gene. Pathotype 1 of race 2 breaks resistance and reproduces on S. bulbocastanum (2). We have tested resistant breeding lines repeatedly in Prosser, WA field plots infested with MC race 1 and harvested tubers free from M. chitwoodi damage. In 2004 however, tubers of some resistant lines were damaged by the M. chitwoodi population that did not cause damage in the past. Populations of M. chitwoodi were established on tomato by adding peels obtained from the infected tubers of resistant lines. The reproductive factor, final number of eggs ÷ initial inoculum, of the new population was determined on five replications of 3-week-old Chantenay carrot and Thor alfalfa. Five thousand eggs were extracted from nematode cultures reared on tomatoes and then were added around the root system of the test plants. The plants were maintained in the greenhouse for 55 days before the nematode eggs were extracted and RF (reproductive factor = final/initial population) values determined. Like the MC race 1, new populations reproduced on Chantenay carrot (RF > 1) but failed to reproduce on Thor alfalfa (RF < 0.1). Unlike MC race 1, the new populations reproduced on roots of all breeding lines that carried Rmc1(blb) gene (RF > 1). These results suggest that the selected population of M. chitwoodi in the Prosser site is a new pathotype, which is designated pathotype 1 of MC race 1. References: (1) C. R. Brown et al. Am. J. Potato Res. 83:1, 2006. (2) H. Mojtahedi et al. J. Nematol. 30:506, 1998. (3) G. S. Santo et al. Plant Dis. 69:361, 1985.

Control of the Oriental Fruit Moth, Grapholita molesta, Using Entomopathogenic Nematodes in Laboratory and Fruit Bin Assays.

The oriental fruit moth (OFM), Grapholita molesta (Busck), which is among the most important insect pests of peaches and nectarines, has developed resistance to a wide range of insecticides. We investigated the ability of the entomopathogenic nematodes (EPN) Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), S. riobrave (Cabanillas et al.), and Heterorhabditis marelatus (Liu and Berry) to control OFM under laboratory and fruit bin conditions. At a dosage of 10 infective juveniles (IJ)/cm(2) in the laboratory, S. carpocapsae caused 63%, S. feltiae 87.8%, S. riobrave 75.6%, and H. marelatus 67.1% OFM mortality. All four nematode species caused significant OFM larval mortality in comparison to the nontreated controls. Steinernema feltiae was used for the bin assays due to the higher OFM mortality it caused than the other tested EPN species and to its ability to find OFM under cryptic environments. Diapausing cocooned OFM larvae in miniature fruit bins were susceptible to IJ of S. feltiae in infested corner supports and cardboard strips. Treatment of bins with suspensions of 10 or 25 S. feltiae IJ/ml water with wetting agent (Silwet L77) resulted in 33.3 to 59% and 77.7 to 81.6% OFM mortality in corner supports and cardboard strips, respectively. This paper presents new information on the use of EPN, specifically S. feltiae, as nonchemical means of OFM control.

First Report of the Stubby-Root Nematode, Paratrichodorus teres, from Potato in the Columbia Basin of Washington State.

Stubby-root nematodes of the genus Paratrichodorus are migratory ectoparasites that cause stunted and deformed roots. They also transmit Tobacco rattle virus, the causal agent of corky ringspot disease in potato. Thus, they are important to the potato industry in the Pacific Northwest, including the Columbia Basin of Washington state, which is one of the largest potato-producing regions in the United States. Stubby-root nematode species are difficult to identify because the morphological differences between the species are very small (4). Paratrichodorus teres is a morphologically similar species and has been reported mainly from temperate regions within Europe. P. teres has also been reported in potato fields in eastern Oregon (3). The morphological similarity between P. allius and P. teres creates problems for routine identification because trichodorid species have a conservative morphology and are separated by the following primary characteristics: females, type of genital system and vulva position; males, number of ventromedian precloacal supplements and cervical papillae and habitus (2). As with most soil nematodes, females are more prevalent than the males. Unfortunately, in the generic key (2), female P. allius and P. teres belong to subgroup 1-12 and are separated by a secondary characteristic, vaginal shape that could be affected by fixation or handling. Male P. allius and P. teres are separated by the former species having one fewer ventromedian precloacal supplement. Thus, the morphological similarity between P. allius and P. teres creates problems for routine identification. This might be one of the reasons that P. teres has not been diagnosed in Washington until now. Using nematode morphological diagnostic keys (1,2) and comparing 18S rDNA sequence data obtained from specimens with those listed on public sequence databases, e.g., NCBI GenBank, stubby-root nematodes from two potato fields close to Moses Lake, Grant County, WA, were diagnosed as P. teres. The two adjacent potato fields are irrigated and are situated approximately 100 miles from the eastern Oregon site where P. teres was first described (3). Soil samples were collected from several Columbia Basin potato fields that had corky ringspot disease and stubby-root nematodes. Corky ringspot disease is an emerging disease in Washington, and the presence of a new viruliferous nematode species might be of concern to the potato industry. References: (1) W. Decraemer. Page 615 in: Manual of Agricultural Nematology. W. R. Nickle, ed. Marcel Dekker, Inc., New York, 1991. (2) W. Decraemer and P. Baujard. Fundam. Appl. Nematol. 21:37, 1998. (3) H. J. Jensen et al. Plant Dis. Rep. 58:269, 1974. (4) S. MacFarlane et al. Adv. Bot. Res. 36:169, 2002.

First Report of Stunting and Root Rot of Potato Associated with Pratylenchus penetrans in the Columbia Basin of Washington.

The root-lesion nematode, Pratylenchus penetrans (de Man, 1880) Filipjev, 1936, is a common pathogen of potato in the United States east of the Rocky Mountains and frequently interacts with Verticillium dahliae to aggravate early dying disease (4). West of the Rocky Mountains, P. penetrans is associated with numerous crops including mint, tree, and small fruits but is rarely recovered from potato fields. Pratylenchus neglectus is a common plant-parasitic nematode on potato in the west, but causes little loss in yield and does not usually interact with V. dahliae (1). Management of P. neglectus is generally unnecessary; although a population of P. neglectus from Ontario, Canada appears to be more pathogenic and does interact with V. dahliae (1). During May 2003 (6 weeks after planting), large areas of stunted plants were observed in field inspections and in aerial photographs of two fields (50.6 ha) of cv. Ranger Russet in Benton County, Washington. Lower roots and stolons had numerous, dark lesions that are typical of P. penetrans damage and were severely stunted, while long, white roots had formed abnormally near the soil surface. In early May 2003, lesion nematodes (65 nematodes per 250 g of dry soil and 810 nematodes per g of fresh root weight) recovered from these potato fields were identified as P. penetrans on the basis of morphological characters (2,3). The crop responded to oxamyl (four applications at 1.1 kg of a.i. per ha between early May and mid-July), but the grower estimated that yields were 1.62 tons/ha (4 tons/acre) less than in comparable unaffected fields. To our knoweldge, this is the first report of severe damage to potato from P. penetrans in the Colombia Basin potato-production area. Soil fumigation with Telone II (1,3-dichloropropene) is commonly used in the Columbia Basin to control root-knot (Meloidogyne chitwoodi and M. hapla) and stubby-root (Paratrichodorus allius) nematodes, and metam sodium is used to control V. dahliae. However, since the only nematode recovered from preplant samples was assumed to be P. neglectus, and because cv. Ranger Russet is relatively tolerant to V. dahliae, no fumigant was used in these fields. An increase in mint production in this area may be responsible for introducing P. penetrans into previously uninfested fields since mint is propagated vegetatively and lesion nematodes are commonly associated with mint and could be easily spread in planting material and adhering soil. Identification of P. penetrans in stunted corn from two nearby fields during 2004 suggests that this nematode may be a new and emerging problem in this area. The Columbia Basin is one of the largest potato-producing regions in the United States, and widespread introduction of P. penetrans could add substantial cost to potato production in this area. References: (1) K. Mukerji. No. 458 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1975. (2) H. Scheck and S. Koike. Plant Dis. 83:877, 1999.

Posttraumatic stress reactions among children following the Athens earthquake of September 1999.

The objective of the present study was to assess symptoms of posttraumatic stress disorder (PTSD), depression and anxiety among children 6 months after they had been exposed to an earthquake (EQ) affecting the northwestern suburbs of Athens in September 1999. A total of 115 children attending two elementary schools located at the epicentre of the EQ were assessed. A group of 48 children not affected by the EQ attending a school not affected by the EQ were used as controls. The children and their parents completed a number of questionnaires. Overall, there was a high rate (78%) of severe to mild PTSD symptoms in the EQ exposed group. Additionally, a substantial proportion of these children scored above criteria (32%) for depression compared to the control group (12.5%). Severe or moderate symptoms of PTSD were associated with high scores of depression (p = 0.002). The relationship between PTSD symptoms and anxiety was limited to the "avoidance" factor of the anxiety questionnaire (p = 0.029). Those who were most likely to be affected were children alone at the time of the EQ, and children who sustained injuries. In summary, countries where EQs are frequent should be prepared to offer psychological support to a substantial proportion of children presenting with PTSD and depressive symptoms and should educate and prepare children to cope with these events.

An Algorithm for Optimizing Rotational Control of Globodera rostochiensis on Potato Crops in Bolivia.

The outline area of new cysts of Globodera rostochiensis was measured by image analysis. A linear regression of this value against egg content provided a basis for adjusting the egg number for cyst size. This adjusted egg content provides an estimate of the relative fullness of a cyst with eggs. This value showed an exponential decline in eggs over 3.5 years since the last potato crop. It corresponds to an average loss in the dormant egg population of 32.8 +/- 5.6%/year for 26 fields at Toralapa, Bolivia. This value compared well with a mean decline of 40 +/- 4%/year for 42 fields after measuring viable eggs/100 g soil on two occasions one year apart. The new approach allows declines to be estimated at one time point. The decline in lipid content of the dormant, unhatched second-stage juveniles (J2) was 17 +/- 6% per annum as measured by image analysis after Oil red O staining. This may be sufficient to compromise infectivity after 3 to 4 years of dormancy. A standard model was modified to consider the effect of both lipid depletion during dormancy and choice of susceptible potato on the population dynamics of G. rostochiensis under rotational control. It is concluded that a few cultivars may impose lower populations on G. rostochiensis in 3 to 4-year rotations than the majority used in Bolivia.

Seasonal Changes in the Dorsal Pharyngeal Gland Nucleolus of Unhatched Second-Stage Juveniles of Globodera spp. in Bolivia.

Changes in the diameter of the nucleolus of the dorsal pharyngeal gland (DPGN) in unhatched second-stage juveniles (J2) of potato cyst nematodes, Globodera spp., were monitored for cysts recovered during two field experiments in the Bolivian Central Andes. In the first experiment, cysts were extracted from soil left fallow or supporting crops of potato, barley, lupin, or quinoa. The highest mean DPGN diameter for unhatched J2 occurred shortly after planting in January. The values were similar for individuals recovered from cysts associated with all cultivations. For cysts from potato plots, the lowest mean DPGN diameter of 2.26 +/- 0.05 mum occurred in March, but the value increased again by May to 2.53 +/- 0.05 mum. Similar seasonal changes were found for J2 under both nonhost crops and fallow with the smallest diameters recorded in May of 2.48 +/- 0.02 mum and 2.34 +/- 0.05 mum, respectively. Two possible factors might cause this significant seasonal change. First, some J2 may hatch early in the growing season, even in the absence of the host. This would enhance the proportion of dormant, unhatched J2 remaining in the cyst samples. Secondly, a seasonal change in the DPGN diameter may occur for most individuals with a transient fall value between January and March/May. A model defined by this study provides a good description of the observed effect, providing both factors are assumed to occur. The second experiment studied if changes in size of DPGN in response to a hatching stimulus are influenced by the cyst population age. The DPGN in unhatched J2 was measured for cysts recovered from soils that had supported potatoes that growing season or 2 or 4 years earlier. The unhatched J2 from the freshly cropped potato site showed the largest mean DPGN diameter of 3.66 +/- 0.05 mum after 7 days in potato root diffusate, whereas those from the 4-year sample had the smallest value of 3.20 +/- 0.05 mum. This significant difference may indicate a delayed response to the hatching stimulus with more prolonged J2 dormancy.

Electrophysiological analysis of the response of males of the potato cyst nematode, Globodera rostochiensis, to fractions of their homospecific sex pheromone.

Electrophysiological techniques and behavioural assays were used to determine the response of males of Globodera rostochiensis to 4 fractions of the homospecific sex pheromone from virgin females, separated using reverse-phase high-pressure liquid chromatography. Sex pheromone activity was confined to 2 of the fractions. The sex pheromone of G. rostochiensis appears to be composed of several weakly basic, polar compounds.

Biochemical analyses on single amphidial glands, excretory-secretory gland cells, pharyngeal glands and their secretions from the avian nematode Syngamus trachea.

The gape nematode, Syngamus trachea, has been used as a model to study nematode secretions. Individual and intact pairs of amphidial glands, pharyngeal glands and pairs of excretory-secretory gland cells have been dissected and their secretory products analysed. The protein profiles of each gland and the total nematode secretions were analysed on 12.5% homogeneous SDS-PAGE minigels. The protein analyses revealed that the structural protein profile of each gland is different. The amphidial gland secretes two major proteins of 36.0 and 41.5 kDa, the excretory-secretory gland cell secretes a protein of 28.2 kDa and a protein of 14.3 kDa, and the pharyngeal gland secretes proteins of 41.5 and 14.6 kDa. Analysis of the total nematode secretions revealed all of the above major secretory proteins and an additional protein of 49.3 kDa. Syngamus trachea secretes acetylcholinesterases and its secretions contain multiple proteases.

Investigation of the chemosensory function of amphids of Syngamus trachea using electrophysiological techniques.

Syngamus trachea, the gape nematode, has been used as a model to study the chemosensory function of amphids. Extracellular electrophysiological recordings were performed directly on amphids. The amphids were stimulated by blood serum from a host bird and by D-tryptophan. The spike frequency produced by the amphid increased significantly after the application of the serum and the application of D-tryptophan. Two types of amphidial neurones responded to the presence of the stimuli. These extracellular recordings have verified the chemosensory function of the amphids.

Temperature-mediated Behavioral Relationships in Bursaphelenchus xylophilus, B. mucronatus, and Their Hybrids.

The influence of temperature on reproduction and movement was examined for seven geographic isolates of Bursaphelenchus xylophilus, three of B. mucronatus, and two of their interspecific hybrids. All nematode isolates tended to be more active and fecund the higher the temperature, with the isolates of B. xylophilus reaching a reproductive peak at higher temperatures than isolates of B. mucronatus. Most isolates of B. xylophilus and B. mucronatus did not produce significantly more progeny at higher male-to-female ratios. The interspecific hybrids appear to possess temperature-related characteristics of either B. xylophilus or both of the parents.

Cryopreservation of the Pinewood Nematode, Bursaphelenchus spp.

Populations of three isolates of Bursaphelenchus xylophilus, the pinewood nematode, and one of B. mucronatus were treated with three cryoprotectants at -70 C for 24 hours followed by deep freezing at -180 C in liquid nitrogen for different periods of time. A solution of 15% glycerol, 35% buffer S, and 50% M9, or 1% aqueous solution of dimethylsulfoxide (DMSO), or a mixture of 60% M9 and 40% S buffer were used as cryoprotectants. A significantly larger number of juveniles than adults survived deep freezing. Significantly more nematodes were motile after cryopreservation in the 15% glycerol-S-M9 soludon than in the M9-S buffer solution or the DMSO aqueous solution. When cryopreserved nematodes that had been treated with glycerol solution were plated onto Botrytis cinerea, they reproduced rapidly over several generations. Cryopreserved nematodes were as pathogenic as untreated nematodes to Scots pines.