Temperature Effects on the Transmission of Bursaphelenchus xylophilus (Nemata: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae)

The object of this research was to investigate the effects of ambient temperature on the transmission of the pinewood nematode, Bursaphelenchus xylophilus, by its vector, Monochamus alternatus. Ninety M. alternatus were reared individually at one of three constant temperatures (16, 20, and 25 °C). As the ambient temperature decreased from 25 to 16 °C, longevity of vectors decreased, nematode transmission efficiency decreased, and the peak period of nematode transmission was delayed and its peak height decreased. Low temperature may inhibit the transmission process, and this inhibition could be partially responsible for preventing pine wilt disease from devastating pine forests in cool regions.     

Effect of Copper Sulfate and Lead Acetate on Infection of Pines with Bursaphelenchus xylophilus

Treatment of 3-year-old Scots, white, and Austrian pine seedlings with copper sulfate or lead acetate significantly affected energy homeostasis and oleoresin production in the seedlings but did not induce wilting of the seedlings. Inoculation of copper sulfate-treated or lead acetate-treated white, Scots, and Austrian pine seedlings with the white pine specific pathotype of Bursaphelenchus xylophilus, VPSt-1, caused a significant increase in oleoresin production, stressed energy homeostasis, and induced rapid wilting of the seedlings. Scots pine lost tolerance and Austrian pine lost resistance to VPSt-1 after the seedlings were treated with either copper sulfate or lead acetate. These results suggest that environmental pollution may significantly affect susceptibility of pines to B. xylophilus and may have a role in establishment of this nematode in uninfested areas.     

Role of Phytotoxins in Pine Wilt Diseases

Characteristic rapid death of pines after infection by Bursaphelenchus xylophilus suggests the involvement of phytotoxins in the pine wilt disease syndrome. Crude extract from diseased pine is toxic to pine seedlings, whereas an extract from healthy pine is not. The response of seedlings to the crude toxin is more prominent in susceptible pine species than in resistant ones. Benzoic acid, catechol, dihydroconiferyl alcohol, 8-hydroxycarvotanacetone (carvone hydrate), and 10-hydroxyverbenone, which are toxic, low molecular weight metabolites, can be isolated from diseased pines. Other unidentified toxins are also found. The toxicity of some of these metabolites correlates positively to the susceptibility of pines to B. xylophilus. Some of these abnormal metabolites show synergistic toxicity when in combination. The D-isomer of 8-hydroxycarvotanacetone, dihydroconiferylalcohol, and 10-hydroxyverbenone inhibited the reproduction of B. xylophilus. Cellulase excreted by pinewood nematode also may be involved in rapid wilting.     

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.     

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.     

Carbon Partitioning in Soybean Infected with Meloidogyne incognita and M. javanica

Seven-day-old seedlings of two cultivars (Cristalina and UFV ITM1) of Glycine max were inoculated with 0, 3,000, 9,000, or 27,000 eggs of Meloidogyne incognita race 3 or M. javanica and maintained in a greenhouse. Thirty days later, plants were exposed to ¹⁴CO₂ for 4 hours. Twenty hours after ¹⁴CO₂ exposure, the root fresh weight, leaf dry weight, nematode eggs per gram of root, total and specific radioactivity of carbohydrates in roots, and root carbohydrate content were evaluated. Meloidogyne javanica produced more eggs than M. incognita on both varieties. A general increase in root weight and a decrease in leaf weight with increased inoculum levels were observed. Gall tissue appeared to account for most of the root mass increase in seedlings infected with M. javanica. For both nematodes there was an increase of total radioactivity in the root system with increased levels of nematodes, and this was positively related to the number of eggs per gram fresh weight and to the root fresh weight, but negatively related to leaf dry weight. In most cases, specific radioactivities of sucrose and reducing sugars were also increased with increased inoculum levels. Highest specific radioactivities were observed with reducing sugars. Although significant changes were not observed in endogenous levels of carbohydrates, sucrose content was higher than reducing sugars. The data show that nematodes are strong metabolic sinks and significantly change the carbon distribution pattern in infected soybean plants. Carbon partitioning in plants infected with nematodes may vary with the nematode genotype.     

Effects of Temperature on Development of Heterodera glycines on Glycine max and Phaseolus vulgaris

Soybean cyst nematode resistant ''Fayette'' and susceptible ''Williams 79'' soybeans (Glycine max) and resistant ''WIS (RRR) 36'' and susceptible ''Eagle'' snap beans (Phaseolus vulgaris) were used in determining the effects of host and temperature on the development, female production, sex ratios, and host response to Heterodera glycines. Temperatures were maintained constant at 16, 20, 24, 28, and 32 C using water-filled tanks. The most rapid development and greatest female production occurred between 20 and 28 C. The equation DS = 5(10⁻⁶)x²y² - 3(10⁻⁴)x²y - 2.8(10⁻³)x² - 1.94(10⁻²)y² + 0.4288x + 1.0220y - 12.7185, where DS = developmental stage, X = time, and Y = temperature, predicted the developmental stage of the nematode and accounted for 84% of the variation. Male : female ratios did not differ within this range and were generally less than one. At all temperatures the resistant soybean produced the greatest number of necrotic responses to H. glycines infection, followed by the resistant snap bean. The susceptible soybean and snap bean produced the fewest necrotic responses.     

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.     

Change in Water Status of Pinus thunbergii Parl. Inoculated with Species of Bursaphelenchus

Maximum and minimum xylem pressure potentials of needles were measured to evaluate water status of Pinus thunbergii Parl. after inoculation with the virulent or avirulent populations of Bursaphelenchus xylophilus or B. mucronatus. In virulent B. xylophilus-inoculated pines, the water status changed abruptly and needle chlorosis occurred by day 29 after inoculation. Similar changes were not seen in B. mucronatus-inoculated and uninoculated control pines. Oleoresin flow ceased in virulent B. xylophilus-inoculated pines. Avirulent B. xylophilus-inoculated pines responded very little to nematode invasion by a slight decrease in oleoresin flow. Oleoresin flow did not vary in B. mucronatus-inoculated and uninoculated control pines. A decrease in soil water potential below field capacity seemed to accelerate the development of pine wilt disease.     

Degree-day Models for Predicting Egg Hatch and Population Increase of Criconemella xenoplax

A degree-day model was derived to predict egg hatch for Criconemella xenoplax. Eggs collected from gravid females were incubated in distilled water at constant temperatures of 10-35 C. Sixty-six percent of all eggs hatched between 13 and 32 C, and 42% hatched at 10 C. All eggs aborted above 32.5 C. Between 25 and 32 C, 8.5 ± 0.5 days were required for egg hatch. Degree-day requirement for egg hatch at 10-30 C was estimated to be 154 ± 5 with a base of 9.03 ± 0.04 C. This base of 9 C was adopted in studies of the relationship between degree-days and nematode population increase on Prunus seedlings grown 9-11 weeks in a greenhouse. Degree-day accumulations were based upon daily averages from maximum and minimum air temperatures. Ratios of final to initial population densities exhibited an exponential pattern in relation to degree-day accumulations with proportionate doubling increment of 0.100 ± 0.049 every 139 ± 8 degree-days. These results provide a means of predicting nematode population increase under greenhouse conditions and a basis for choosing sampling intervals when evaluating nematode multiplication.     

Ditylenchus dipsaci Infestation of Trifolium repens. I. Temperature Effects,Seedling Invasion,and a Field Survey

Rates of development of stem nematode (Ditylenchus dipsaci) in white clover (Trifolium repens) seedlings were found to be linearly related to temperature. Basal developmental temperature (T_b) was 3 °C, and the thermal constant (S) for development of gravid adult females from freshly laid eggs was 270 accumulated day-degrees above the T_b. Only 12% at 20 °C and 4% at 4 °C of the gravid female nematodes inoculated into seedling axils successfully penetrated seedling epidermis. These nematodes slowly migrated within the seedling and after a lag of 5 days at 20 °C started to lay eggs. The maximal rate of egg production was temperature-dependent, being 0.8 and 3.1 eggs female⁻¹ day⁻¹ at 10 and 20 °C, respectively. Nematodes emigrated rapidly from infested stolons when they were immersed in water, with rates being highest at 25 °C and lowest at 4 °C. The sensitivity to temperature of many of the parameters that govern nematode population dynamics indicates that climatic changes will have a marked effect upon this host-parasite system. A study of infested stolons from the field indicated that nematode numbers increased up to 3,000 or more before tissue senesence, triggered by nematode damage, caused a mass emigration of nematodes from the stolon.     

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.     

Uptake of Lipids by the Entomophilic Nematode Romanomermis culicivorax

Romanomermis culicivorax juveniles were dissected out of Aedes aegypti larvae 7 days after infection and incubated under controlled conditions in isotonic saline containing a ¹⁴C-labeled fatty acid (palmitic acid), monoacylglycerol (glycerol monoolein), or triacylglycerol (glycerol tripalmate) nutrient source. The mermithid absorbed each of these lipids from the incubation medium, the rate of uptake being greatest for glycerol monoolein. No lipase activity was detected in whole nematode homogenates or in the media in which the nematodes were incubated. It is suggested that the nematode transports complex lipid molecules across its outer cuticle intact.     

Recent Developments in Nematode Steroid Biochemistry

Current knowledge of steroid nutrition, metabolism, and function in free-living, plant-parasitic and animal-parasitic nematodes is reviewed, with emphasis upon recent investigation of Caenorhabditis elegans. A number of 4-desmethylsterols with a trans-A/B ring configuration can satisfy the steroid nutritional requirement in C. elegans, but sterols with a cis-A/B ring configuration or trans-A/B sterols with a 4-methyl group cannot. C. elegans removes methyl or ethyl substituents at C-24 of the plant sterols sitosterol, campesterol, stigmasterol, stigmastanol, and 24-methylene-cholesterol to produce various sterols with structures partially dependent upon that of the dietary sterol. Additional metabolic steps in C. elegans include reduction of Δ²²- and Δ⁵-bonds, C-7 dehydrogenation, isomerization of a Δ⁷-bond to a Δ⁸⁽¹⁴⁾-bond, and 4α-methylation. An azasteroid and several long-chain alkyl amines interfere with the dealkylation pathway in C. elegans by inhibiting the Δ²⁴-sterol reductase; these compounds also inhibit growth and reproduction in various plant-parasitic and animal-parasitic nematodes. A possible hormonal role for various steroids identified in nematodes is discussed.     

Biological Control of Soil Pests by Mixed Application of Entomopathogenic and Fungivorous Nematodes

In greenhouse experiments, massive application of the fungivorous nematode, Aphelenchus avenae, in summer at 26-33 C (1 x l0⁵ nematodes/500 cm³ autoclaved soil) or in autumn at 18-23 C (5 x 10⁴ nematodes/500 cm³ autoclaved soil) suppressed pre-emergence damping-off of cucumber seedlings due to Rhizoctonia solani AG-4 by 67% or 87%, respectively. Application of 2 x l0⁵ A. avenae to sterilized soil infested with R. solani caused leafminer-like symptom on the cotyledons, which did not occur in mixed inoculations with the entomopathogenic nematode, Steinernema carpocapsae. When 1 x 10⁶ A. avenae were applied 3 days before inoculation with 100 Meloidogyne incognita juveniles, gall numbers on tomato roots were reduced to 50% of controls. Gall numbers also were suppressed by S. carpocapsae (str. All). Reduction in gall numbers was no greater with mixed application of A. avenae and S. carpocapsae than with application of single species, even though twice the number of nematodes were added in the former case. These nematodes were positively attracted to tomato root tips. Aphelenchus avenae suppressed infection of the turnip moth, Agrotis segetum, but not the common cutworm, Spodoptera litura, by S. carpocapsae.     

Distribution and Population Dynamics of Nematodes in a Rice Field and Pasture in India

Ecological studies on soil nematodes were made in a tropical rice field and pasture. Parasitic species were more diversified in the pasture than in the rice field. Eighty-six and sixty percent of total nematodes occurred in the top 10 cm in rice field and pasture, respectively. Nematodes were not randomly or uniformly dispersed but aggregated. Parasitic forms were most abundant and correlated with root biomass in the 0-15-cm soil layer, the greatest number usually occurring at the 10-15-cm depth at both sites. In summer, however, they were densest at the 15-30-cm depth. Microbivores were most frequent in the top 5 cm of both sites. Micellaneous feeders (food sources uncertain) usually occurred in highest densities at the 15-30-cm depth. Predators showed no distinct depth preference. Temperature and moisture of the soil apparently played an important role in regulating nematode population. Peak densities of 31.3 × 10⁴/m² and 21.6 × 10⁴/m² at a 30-cm depth occurred in January, while minimum densities of 5.0-5.3 × 10⁴/m² and 4.1 × 10⁴/m² occurred in July-October and April in rice field and pasture, respectively. Monthly mean biomass of nematodes was 23.8 ± 4.5 mg/m² in rice field and 11.5 ± 1.5 mg/m² in pasture.     

Carbohydrate Concentration in Pine as Affected by Inoculation with Bursaphelenchus xylophilus

Pines responded to inoculation with Bursaphelenchus xylophilus by changes in reducing and nonreducing carbohydrate concentrations dependent on the pine species and the pathotype of B. xylophilus with which the trees were inoculated. Carbohydrate concentrations, in compatible pine-nematode pathotype combinations, decreased initially after inoculation and then increased slightly before decreasing to approximately 10% of the control levels as the seedlings wilted. In compatible nematode pathotype-pine species combinations, carbohydrate concentrations decreased and then increased as the nematode population densities declined.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

Transport of Palmitic Acid Across the Tegument of the Entomophilic Nematode Romanomermis culicivorax

Romanomermis culicivorax juveniles, dissected out of Aedes aegypti larvae 7 days after infection, were incubated under controlled conditions in isotonic saline containing ¹⁴C-U-palmitic acid to investigate the nature of the transport mechanism(s) used by the nematode for transcuticular uptake of palmitic acid. Net uptake of the isotope by the nematode was of a logarithmic nature with respect to time. Uptake of palmitic acid was accomplished by a combination of diffusion and a mediated process which was substrate saturable and competitively inhibited by myristic and stearic acids. Both 2,4-dinitrophenol and ouabain inhibited uptake of palmitic acid and thus supported the hypothesis that the carrier system is of the active transport variety and is coupled to a Na⁺K⁺ ATPase pump.     

Importance of Temperature in the Pathology of Meloidogyne hapla and M. chitwoodi on Legumes

Effects of temperatures on the host-parasite relationships were studied for three legume species and four populations of root-knot nematodes from the western United States. The nematode populations were Meloidogyne hapla from California (MHCA), Utah (MHUT), and Wyoming (MHWY), and a population of M. chitwoodi from Utah (MCUT). The legumes were milkvetch (Astragalus cicer), alfalfa (Medicago sativa), and yellow sweet clover (Melilotus officinalis). All milkvetch plants survived inoculation with all nematode populations, while alfalfa and yellow sweet clover were more susceptible. On yellow sweet clover, MHCA was most pathogenic at 30 °C based on suppression of shoot growth while MHUT, MHWY, and MCUT were most pathogenic at 25 °C. All nematode populations suppressed growth of yellow sweet clover more than growth of milkvetch and alfalfa. The reproductive factor (Rf = final nematode population/initial nematode population) of MHCA was positively correlated (r = 0.83) with temperature between 15 °C and 30 °C. The greatest Rf occurred on alfalfa inoculated with MHCA at 30 °C. The Rf of MHUT, MHWY, and MCUT were positively correlated (r= 0.76, r= 0.78, and r= 0.73, respectively) with temperature between 15 °C and 25 °C. The Rf values of MHUT and MHWY were similar on all species and exceeded the Rf of MCUT at all temperatures (P < 0.05).