Pathogenicity of Bursaphelenchus xylophilus on Pines in Minnesota and Wisconsin

The pinewood nematode, Bursaphelenchus xylophilus, was inoculated into established native jack and red pines (Pinus banksiana and P. resinosa) and exotic Austrian pine (P. nigra) in Minnesota and Wisconsin forests during summer 1981. The nematode isolates did not kill established nonstressed pine trees growing in the forest. However, the same nematode isolates killed pine seedlings under greenhouse conditions. Girdling the main stem of some trees to induce stress resulted in the death of the majority of inoculated and noninoculated branches of Austrian and jack pines, but no branch death was observed on red pine. Greater numbers of nematodes were extracted from branches of inoculated, girdled trees than from nongirdled trees. The mean number of nematodes extracted from branches of inoculated, nongirdled trees was 0.3 - 14 nematodes per gram of wood.     

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.     

Transmission of Pinewood Nematode Through Feeding Wounds of Monochamus carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode, Bursaphelenchus xylophilus, to mature, field grown Scots pines through feeding wounds of Monochamus carolinensis was investigated by caging nematode-infested beetles on pine branches for 24 hours. Nematodes were transmitted to 31 of 64 branches. Frequency of successful transmission was independent of the sex of the beetle but dependent upon beetle age. Transmission frequencies were highest for beetles 2, 3, and 4 weeks after emergence as adults. The number of nematodes transmitted per branch was low and did not differ between beetle sexes or among beetle age categories. The number of nematodes extracted per branch was correlated with the number of nematodes carried per beetle but was not correlated with the feeding area on the branch.     

Survival and Infectivity of Bursaphelenchus xylophilus in Wood Chip-Soil Mixtures

To determine the effect of soil environment on the life stages and total numbers of Bursaphelenchus xylophilus, nematode-infested wood chips alone and mixed with soil were incubated at 12 and 20 C. Nematodes were extracted at 2-week intervals for 12 weeks. Numbers of nematodes and percentage of third-stage dispersal larvae were greater at 12 C and in chips without soil. Percentage of juveniles of the propagative cycle was greater at 20 C and in chips with soil. Although B. xylophilus survived in chips with soil for 12 weeks, nematode numbers and life stage percentages changed little over time. To determine if B. xylophilus was capable of infecting wounded roots, infested and uninfested chips were mixed with soil in pots with white and Scots pine seedlings. Trees were maintained at 20 and 30 C and harvested at mortality or after 12 weeks. Only seedlings treated with infested chips contained nematodes. In field experiments, planted seedlings were mulched with infested chips to determine if nematodes would invade basal stem wounds. Among these trees, Scots pine was more susceptible than white or red pines to infection and mortality.     

Transmission of Bursaphelenchus xylophilus through Oviposition Wounds of Monochamm carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode through Monochamus carolinensis oviposition wounds was documented. Nematode transmission was measured as the average number of nematodes isolated per oviposition wound excavated and also as the percentage of oviposition wounds from which nematodes were isolated. The influence of three factors that might affect nematode transmission was investigated: age of the beetle vector, number of nematodes carried per beetle, and egg deposition in the oviposition wound. Only the number of nematodes carried by the beetle was found to have a significant effect on transmission. Nematodes were transmitted more frequently and in slightly greater numbers by beetles carrying more nematodes. The influence of pinewood on nematode exit from beetles were investigated by comparing nematode exit from beetles placed over pine chips with those placed over distilled water. Nematodes exited in greater numbers and at a higher frequency from beetles over pine chips than from beetles over distilled water. Apparently, the nematodes are able to detect a factor from the pine chips that promotes their exit from the beetles.     

Nemtaode-Vector Relationships in the Pine Wilt Disease System

Pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease in North America and Japan. Dispersal stage dauer larvae are transported to new host trees on the body surface and within the tracheal system of several beetle species. Worldwide, 21 species of Cerambycidae, 1 genus of Buprestidae, and 2 species of Curculionidae are known to carry pinewood nematode dauer larvae upon emerging from nematode-infested trees. Five species of cerambycids in the genus Monochamus are known to transmit dauer larvae to new host trees, four North American species and one Japanese species. Primary transmission to healthy trees occurs through beetle feeding wounds on young branches. Secondary transmission to stressed trees or recently cut logs occurs through Monochamus oviposition sites.     

Population Development and Influence of Bursaphelenchus xylophilus on Gliocladium virens

Gliocladium virens was isolated from slash pine trees symptomatic and asymptomatic for pine wilt disease with frequencies of 24% and 10%, respectively. Populations of Bursaphelenchus xylophilus, the nematode incitant of this disease, reproduced on this fungus and inhibited its growth. Growth inhibition of the fungus was characterized by an absence of sporulation and by the formation of chains of dark, thick-walled, chlamydospore-like cells. Population increase during a 12-day period following infestation of cultures of the fungus with 10,000 nematodes averaged 3-fold at 16 C, 9-fold at 20 C, and 24-fold at 24 C. In greenhouse studies, nematode recovery from slash pine seedlings coinoculated with both organisms was significantly greater than that obtained from seedlings inoculated with the nematode alone.     

Pinewood Nematode,Bursaphelenchus xylophilus,Associated with Red Pine, Pinus resinosa,in Western Maryland

Red pines Pinus resinosa in Garrett and Allegany counties, Maryland, were examined during 1982-84 to determine distribution of the pinewood nematode, Bursaphelenchus xylophilus, within and among trees. Approximately 25-year-old (younger) and 47-year-old (older) trees were subdivided into the following categories: 1) trees with mostly green needles; 2) trees with mostly reddish-brown needles; 3) trees lacking needles but with bark intact; 4) trees lacking both needles and bark; and 5) trees with chlorotic, bleached-green needles. Bursaphelenchus xylophilus was found infecting 68% of younger red pines and 77% of older red pines. Nematodes were not evenly distributed in trees within any given tree decadence category or in trees of the same age. Nematodes were recovered from 20% of wood samples from trunks and primary and secondary branches in younger pines and from 15 % of older red pines. On the basis of tree decadence category, the highest incidence of infection in younger trees (31%) was in bleached-green needled trees (category 5), whereas in older trees the highest infection (25%) occurred in green needled trees (category 1). At both sites trunks were infected more often than branches.     

Within-wood Spatial Dispersion of the Pinewood Nematode,Bursaphelenchus xylophilus

Pinewood nematode, Bursaphelenchus xylophilus (Steiner &Buhrer) Nickle, spatial dispersion was determined in Scots pine, Pinus sylvestris L., bolts infested with the pine sawyer beetle, Monochamus carolinensis (Olivier) and in bolts without M. carolinensis. According to Taylor''s power law and Green''s index of dispersion, nematode dispersion was aggregated in both sets of bolts. The degree of aggregation did not differ significantly between beetle-infested and noninfested bolts, suggesting that the presence of M. carolinensis does not affect nematode dispersion within a bolt. Nematode population densities differed radially in bolts not infested with pine sawyers, but in a nonregular pattern. Moisture content of the bolts was correlated with population density of B. xylophilus, suggesting that nematode aggregates occur in areas of high moisture content.     

Protocylindrocorpus dendrophilus n. sp. (Nematoda: Cylindrocorpidae) Associated with Pine Wood Borings

Protocylindrocorpus dendrophilus n. sp. is described from xylem samples taken from beetle infested slash pine (Pinus elliottii Engelmann var. elliottii) in Central Louisiana. It is similar to P. goodeyi (Rühm) Paramonov, but differs by the possession of a protuberant and more posteriorly located vulva and in the position of the caudal papillae. Morphometrics of the male and female are presented.     

Pathogenesis in Pine Wilt Caused by Pinewood Nematode, Bursaphelenchus xylophilus

The progression of events in the development of pine wilt disease following the invasion by Bursaphelenchus xylophilus is reviewed from early migration through pine tissues until symptom development on foliage. Disease resistance in pines, especially the hypersensitive reaction that is successful in controlling many potential pests and pathogens, is explored. Pathologies resulting from the activities of pinewood nematode include cortical trails and cavities; formation of cambial gaps and traumatic resin cysts; browning and death of cortex, phloem, cambium, and ray tissues; granulation and shrinkage of cell cytoplasm in rays; and destruction of resin canal epithelial and ray parenchyma cells. Death of parenchyma, production of toxins, and leakage of oleoresins and other material into tracheids are typical of the hypersensitive reaction occurring in pines following migration of small numbers of pinewood nematodes. The hypothesis presented is that a spreading hypersensitive reaction results in some of the observed pathologies and symptoms and eventually causes pine death. The growth-differentiation balance hypothesis is used to help explain predisposition, oleoresin production and toxicity, susceptibility and resistance, and the effects of variation in climate on host pines as related to pinewilt disease.     

A Nondestructive Method of Determining Bursaphelenchus xylophilus Infestation of Monochamus spp. Vectors

Pine wilt is caused by the nematode Bursaphelenchus xylophilus, which is transported to host trees in the trachea of Monochamus spp. (Coleoptera: Cerambycidae). The study of the relationship between the nematode and its beetle vectors has been hampered by the inability to estimate nematode presence or density within live beetles. This report describes a rapid method for estimating nematode load within live M. carolinensis and M. alternatus by visual examination of the atrium of the first abdominal spiracle. Visual estimates of nematode numbers correlated highly with actual nematode numbers. This method is a timesaving technique for determining relative numbers of B. xylophilus in pine wilt research.     

Relative Susceptibility of Four Pine Species to Infection by Pinewood Nematode

Mature trees of eastern white, jack, Scotch, and shortleaf pines were inoculated with 25,000-34,000 pinewood nematodes, Bursaphelenchus xylophilus, isolated from infected Scotch pines in Missouri. Equal numbers of trees of each species inoculated with distilled water served as controls. Nine of fifteen Scotch pines died within 4 months of nematode infection or during the winter and early spring following infection. A single eastern white and shortleaf pine died. No jack pines died. A single Scotch pine control died, apparently the result of natural nematode infection. No other controls died. Mean oleoresin flow did not differ among nematode-inoculated jack and shortleaf pines and their respective controls. Oleoresin flow in nematode-inoculated eastern white and Scotch pines was significantly lower than in their controls. Oleoresin flow was temporarily reduced in mortality-resistant eastern white and Scotch pines following nematode infection. Thus a sublethal impact of nematode infection on mortality-resistant host trees was documented.     

Host Status of Five Weed Species and Their Effects on Pratylenchus zeae Infestation of Maize

The host suitability of five of the most common weed species occurring in maize (Zea mays L.) fields in South Africa to Pratylenchus zeae was tested. Based on the number of nematodes per root unit, mealie crotalaria (Crotalaria sphaerocarpa) was a good host; goose grass (Eleusine indica), common pigweed (Amaranthus hybridus), and thorn apple (Datura stramonium) were moderate hosts; and khaki weed (Tagetes minuta) was a poor host. Only the root residues of khaki weed suppressed the P. zeae infestation of subsequently grown maize. When goose grass, khaki weed, and mealie crotalaria were grown in association with maize in soil infested with P. zeae, goose grass and khaki weed severely suppressed maize root development; this resulted in a low number of nematodes per maize root system and a high number of nematodes per maize root unit. Mealie crotalaria did not restrict maize root growth and did not affect nematode densities per maize root system or maize root unit. Special attention should be given to the control of mealie crotalaria, which is a good host for P. zeae, and goose grass, which, in addition to its ability to compete with maize, is also a suitable host for P. zeae.     

Observations on the Morphology of the Pine Wood Nematode, Bursaphelenchus xylophilus

Scans of slash pine wood chips infested with pine wood nematodes showed coiled aggregates of 5-10 nematodes in the axial resin canals, and 1-2 nematodes folded in the radial resin canals. Observations of the pine wood nematode by SEM showed the head to be offset with six labial lobes. A smaller circle of six inner lobes surrounded the stoma. A single labial sensillum was observed on each of the two subventral and two subdorsal lobes. The two opposite lobes, without sensilla, bore a pore-like amphid. The female vulva flap was observed to be a pouch. In addition to two pairs of copulatory papillae previously described for the male, three additional papillae were observed: an additional post-anal pair, and a single papilla anterior to the cloaca. Spicules were fused ventrally. Distal spicule ends terminated in two fused half discs. Caudal alae were angular at the tail terminus, but culed inward at the corners to appear oval. Lateral fields had four lateral striations.     

Temporal Pattern of Pinewood Nematode Exit from the Insect Vector Monochamus carolinensis

Laboratory-reared Monochamus carolinensis (Olivier) were used to study the temporal pattern of pinewood nematode dauer larval exit from this beetle vector. Exit rates of dauer larvae were determined by comparing the mean number of dauer larvae carried by adult beetles 0, 7, 14, or 21 days after emergence from the log in which they developed. Density of dauer larvae was highest in beetles on the day of their emergence and dropped slowly through the subsequent age classes. The rate of nematode exit was low during the first week (4.5%) and higher during weeks 2 (20.5%) and 3 (13.1%). A total of 38.1% of the initial dauer larvae exited the beetles during the 3-week observation period.     

History of Pine Wilt Disease in Japan

Pine wilt disease induced by the pinewood nematode, Bursaphelenchus xylophilus, is a great threat to pine forests in Japan. The first occurrence of the disease was reported in Nagasaki, Kyushu. During the 1930s the disease occurrence was extended in 12 prefectures, and in the 1940s the disease was found in 34 prefectures. The annual loss of pine trees increased from 30,000 m³ to 1.2 million m³ during these two decades. The enormous increase in timber loss in the 1970s resulted in 2.4 million m³ of annual loss in 1979. The affected area expanded into 45 prefectures of 47 prefectures in Japan. In cool areas the disease differs in epidemiology from that in heavily infested areas in the warm regions. A national project for controlling pine wilt disease lays special emphasis on the healthy pine forests predominating throughout cool areas in northern Japan.     

In Vitro Culture and Feeding Behavior of Belonolaimus longicaudatus on Excised Zea mays Roots

A greenhouse population of the sting nematode, Belonolaimus longicaudatus, obtained from an infested golf course in California''s Coachella Valley, was surface-decontaminated and cuhured on excised roots of Zea mays supported by Gamborg''s B5 medium. At 26-27 °C the females laid eggs, and newly emerged juveniles of the second generation completed three molts within 29 days after egg deposition. Sixty days after inoculation with 60 females and 40 males, an average of 529 nematodes and 83 eggs were recovered from the culture. The feeding process consisted of probing, stylet penetration, ingestion, and stylet retraction. Feeding seemed to be necessary before egg deposition or molting occurred. The sting nematode was observed feeding exclusively as an ectoparasite and preferably at the region of cell division and elongation. Vigorous feeding by many nematodes usually caused discoloration of root tips and termination of growth.     

Role of Nematodes,Nematicides, and Crop Rotation on the Productivity and Quality of Potato,Sweet Potato,Peanut, and Grain Sorghum

The objective of this experiment was to determine the effects of fenamiphos 15G and short-cycle potato (PO)-sweet potato (SP) grown continuously and in rotation with peanut (PE)-grain sorghum (GS) on yield, crop quality, and mixed nematode population densities of Meloidogyne arenaria, M. hapla, M. incognita, and Mesocriconema ornatum. Greater root-gall indices and damage by M. hapla and M. incognita occurred on potato than other crops. Most crop yields were higher and root-gall indices lower from fenamiphos-treated plots than untreated plots. The total yield of potato in the PO-SP and PO-SP-PE-GS sequences increased from 1983 to 1985 in plots infested with M. hapla or M. arenaria and M. incognita in combination and decreased in 1986 to 1987 when root-knot nematode populations shifted to M. incognita. The total yields of sweet potato in the PO-SP-PE-GS sequence were similar in 1983 and 1985, and declined each year in the PO-SP sequence as a consequence of M. incognita population density increase in the soil. Yield of peanut from soil infested with M. hapla increased 82% in fenamiphos-treated plots compared to untreated plots. Fenamiphos treatment increased yield of grain sorghum from 5% to 45% over untreated controls. The declining yields of potato and sweet potato observed with both the PO-SP and PO-SP-PE-GS sequences indicate that these crop systems should not be used longer than 3 years in soil infested with M. incognita, M. arenaria, or M. hapla. Under these conditions, these two cropping systems promote a population shift in favor of M. incognita, which is more damaging to potato and sweet potato than M. arenaria and M. hapla.