Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae)

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.     

Spatial pattern of pine wilt disease caused byBursaphelenchus xylophilus (Nematoda: Aphelenchoididae) within aPinus thunbergii stand

To understand the mechanism of spread of pine wilt disease caused by the pinewood nematode, Bursaphelenchus xylophilus, which is vectored by a cerambycid, Monochamus alternatus, the spatial distribution of trees weakened by the nematode was examined within a Pinus thunbergii stand from June to October for 4 years. The weakened trees were distributed in a clumped pattern in 1980 and 1981, at an early stage of infestation. In many cases, they showed a double-clumped pattern. The degree of aggregation was higher in June or July than after August. They were uniformly distributed in June or July 1982 and in June 1983 whereas they showed a double-clumped pattern after August. The trees were frequently weakened in June or July when they were near the trees weakened during the previous year. At quadrat sizes of more than 25 m², spatial overlapping was pronounced between trees weakened during June–July of the current year and those weakened in the previous year. The seasonal changes in spatial distribution of weakened trees were explained by the interaction among M. alternatus, B. xylophilus and Pinus trees.     

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae)

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that “Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad‐leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus.” We used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer‐using Monochamus beetles after transfer by A. fraudatrix from broad‐leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.     

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.     

Nematicidal and Propagation Activities of Thyme Red and White Oil Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

The toxic and propagation effects on Bursaphelenchus xylophilus of 28 Thymus vulgaris red oil and white oil compounds were examined using direct contact and cotton ball bioassays. Results were compared with those of the trunk-injection nematicides emmamectin benzoate, levamisol hydrochloride and morantel tartrate. In direct contact bioassays, geraniol (LC₅₀, 0.47 mg/ml) was the most toxic compound, followed by thymol (1.08 mg/ml), carvacrol (1.23 mg/ml) and terpinen-4-ol (2.61 mg/ml). In cotton ball tests with 20 inactive compounds at 2 mg/cotton ball, p-cymene significantly inhibited propagation (propagation ratio [PR] 8), compared with the castor oil-ethanol-treated control (PR 56). Propagation stimulation was observed with (–)-caryophyllene oxide, (+)-ledene, (+)- and (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene (PR 63–100). The other 10 compounds exhibited low to moderate levels of propagation inhibition (PR 36–56). At 0.1 μg/cotton ball, emmamectin benzoate and morantel tartrate exhibited complete suppression of propagation, whereas a very low level of propagation inhibition was obtained from levamisol hydrochloride (PR 6). In conclusion, propagation-stimulating compounds can exist in plants in addition to nematicidal compounds, and careful use of plant preparations containing high quantities of these compounds is mandatory.     

Nematicidal Activity of Cassia and Cinnamon Oil Compounds and Related Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

The nematicidal activity of two cassia, Cinnamomum cassia, oils (Especial and true), four cinnamon, Cinnamomum zey-lanicum, oils (technical, #500, bark and green leaf), and their compounds (e.g., trans-cinnamaldehyde and trans-cinnamic acid) toward adult Bursaphelenchus xylophilus was examined by a direct contact bioassay. Results were compared with those of 34 related compounds. As judged by 24-hour LC₅₀ values, two cassia oils (0.084–0.085 mg/ml) and four cinnamon oils (0.064–0.113 mg/ml) were toxic toward adult B. xylophilus. Of 45 test compounds, trans-cinnamaldehyde (0.061 mg/ml) was the most active nematicide, followed by ethyl cinnamate, α-methyl-trans-cinnamaldehyde, methyl cinnamate and allyl cinnamate (0.114–0.195 mg/ml). Potent nematicidal activity was also observed with 4-methoxycinnamonitrile, trans-4-methoxycinnamaldehyde, trans-2-methoxy-cinnamaldehyde, ethyl α-cyanocinnamate, cinnamonitrile and cinnamyl bromide (0.224–0.502 mg/ml). Structure-activity relationships indicate that structural characteristics, such as types of functional groups, saturation and carbon skeleton, appear to play a role in determining the toxicities to adult B. xylophilus. Cassia and cinnamon oils and test compounds described merit further study as potential nematicides or leads for the control of pine wilt disease caused by B. xylophilus.     

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster)

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera.The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA.The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines.In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.     

Effectiveness of mass trapping in the reduction of Monochamus galloprovincialis Olivier (Col.: Cerambycidae) populations

Monochamus galloprovincialis Olivier beetles vector the causal agent of pine wilt disease (PWD), nematode Bursaphelenchus xylophilus (Steiner and Bührer) Nickle, in Europe. Traps and attractants have been optimized for the capture of M. galloprovincialis, increasing the possibility of developing methods of lowering its population in PWD‐affected areas with the aim of either eradicating the disease or containing the spread of it. To evaluate the effectiveness of such mass‐trapping campaigns, two sets of experiments were carried out in 2010 and 2013. The release of 353 laboratory‐reared beetles in the experimental area of 2010 facilitated the evaluation of capture–mark–recapture (CMR) procedures in the calculation of population abundance estimates using the POPAN formulation of the Jolly–Seber model, a prerequisite for the assessment of mass trapping. Abundance estimates derived from best‐fitting parameters fell within one standard error of the real figures, proving the method appropriate. In 2013, four trap densities were tested in six 36 ha plots. To evaluate the removed proportions, the local beetle population was estimated in a contiguous 260 ha study area. A superpopulation of 21 319 individuals could be calculated from the CMR data, corresponding to a rough density of 82 individuals per hectare. Evaluated trapping densities removed 4.66%, 20.50%, 33.33% and 59.80% of M. galloprovincialis population at 0.02, 0.11, 0.25 and 0.44 traps/ha, respectively, thus the estimated 95% removal would occur at 0.82 traps/ha. These results suggest that substantial reduction of M. galloprovincialis abundances might be achieved via mass trapping and that this represents a very promising management method for the containment or eventual eradication of B. xylophilus at the areas affected by the PWD.     

Species of Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) and other nematode genera associated with insects from Pinus pinaster in Portugal

Insects associated with maritime pine, Pinus pinaster, in Portugal were collected and screened for the presence of Bursaphelenchus species. Nematodes were identified using Internal Transcribed Spacers‐Restriction Fragment Length Polymorphism (ITS‐RFLP) analysis of dauer juveniles and morphological identification of adults that developed from dauer juveniles on fungal cultures or on cultures in pine wood segments at 26°C. Several associations are described: Bursaphelenchus teratospicularis and Bursaphelenchus sexdentati are associated with Orthotomicus erosus; Bursaphelenchus tusciae, B. sexdentati and/or Bursaphelenchus pinophilus with Hylurgus ligniperda and Bursaphelenchus hellenicus with Tomicus piniperda, Ips sexdentatus and H. ligniperda. An unidentified Bursaphelenchus species is vectored by Hylobius sp. The previously reported association of Bursaphelenchus xylophilus with Monochamus galloprovincialis was confirmed. The association of Bursaphelenchus leoni with Pityogenes sp. is not definitively established and needs further studies for clarification. Other nematode genera besides Bursaphelenchus were found to be associated with the insects sampled, including two different species of Ektaphelenchus, Parasitorhabditis sp., Parasitaphelenchus sp., Contortylenchus sp. and other unidentified nematodes. The Ektaphelenchus species found in O. erosus is morphologically similar to B. teratospicularis found in the same insect; adults of both the species are found in cocoon‐like structures under the elytra of the insects.     

Cloning and characterization of a 2-cys peroxiredoxin in the pine wood nematode, Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation

The pine wood nematode, Bursaphelenchus xylophilus, is an invasive plant parasitic nematode and a worldwide quarantine pest. An indigenous species in North America and the causal agent of pine wilt disease, B. xylophilus has devastated pine production in Southeastern Asia including Japan, China, and Korea since its initial introduction in the early 1900s. The reactive oxygen species (ROS) is the first line of defense utilized by host plants against parasites, while nematodes, counteractively, employ antioxidants to facilitate their infection. Peroxiredoxins (Prxs) are a large class of antioxidants recently found in a wide variety of organisms. In this report, a gene encoding a novel 2-cysteine peroxiredoxin protein in B. xylophilus was cloned and characterized. The 2-cysteine peroxiredoxin in B. xylophilus (herein refers to as "BxPrx") is highly conserved in comparison to 2-cysteine peroxiredoxins (Prx2s) in other nematodes, which have two conserved cysteine amino acids (Cp and Cr), a threonine-cysteine-arginine catalytic triad, and two signature motifs (GGLG and YF) sensitive to hydrogen peroxide. In silico assembly of BxPrx tertiary structure reveals the spatial configuration of these conserved domains and the simulated BxPrx 3-dimensional structure is congruent with its presumed redox functions. Although no signal peptide was identified, BxPrx was abundantly expressed and secreted under the B. xylophilus cuticle. Upon further analysis of this leader-less peptide, a single transmembrane α-helix composed of 23 consecutive hydrophobic amino acids was found in the primary structure of BxPrx. This transmembrane region and/or readily available ATP binding cassette transporters may facilitate the transport of non-classical BxPrx across the cell membrane. Recombinant BxPrx showed peroxidase activity in vitro reducing hydrogen peroxide using glutathione as the electron donor. The combined results from gene discovery, protein expression and distribution profiling (especially the "surprising" presence under the nematode cuticle), and recombinant antioxidant activity suggest that BxPrx plays a key role in combating the oxidative burst engineered by the ROS defense system in host plants during the infection process. In summary, BxPrx is a genetic factor potentially facilitating B. xylophilus infestation.     

Trapping strategy for Monochamus sutor and Monochamus galloprovincialis: potential vectors of the pine wood nematode in Scandinavia

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Bursaphelenchus anatolius n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Bees in the Genus Halictus

Bursaphelenchus anatolius n. sp., a phoretic associate of Halictus bees from Ankara, Turkey, is described and illustrated. Bursaphelenchus anatolius n. sp. is closest to B. kevini, which is phoretically associated with Halictus bees from the Pacific Northwest. Bursaphelenchus anatolius n. sp. and B. kevini appear to be sister taxa based upon several shared morphological features, similar life histories involving phoresy with soil-dwelling Halictus bees, and molecular analysis of the near-full-length small subunit rDNA, D2D3 expansion segments of the large subunit rDNA, and partial mitochondrial DNA COI. Bursaphelenchus anatolius n. sp. can be differentiated from all other species of Bursaphelenchus based upon spicule morphology. The paired spicules are uniquely shaped and ventrally recurved, and both B. anatolius n. sp. and B. kevini possess extending flaps that open when the spicules are protracted beyond the cloaca. Population growth of B. anatolius n. sp. was measured at 23 °C in the laboratory on cultures of the fungus Monilinia fructicola grown on lactic acid-treated, 5% glycerol-supplemented potato dextrose agar. Nematode population densities rapidly increased from 110 to about 110,000/9-cm-diam. dish within 21 days.     

Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muricata)

The ectomycorrhizal fungal associations of Douglas fir ( Pseudotsuga menziesii D. Don) and bishop pine ( Pinus muricata D. Don) were investigated in a mixed forest stand. We identified fungi directly from field-collected ectomycorrhizal (ECM) root tips using PCR-based methods. Sixteen species of fungi were found, of which twelve associated with both hosts. Rhizopogon parksii Smith was specific to Douglas fir. Three other species colonized only one of the hosts, but were too infrequent to draw conclusions about specificity. Seventy-four percent of the biomass of ECM root tips sampled in the stand were colonized by members of the Thelephoraceae and Russulaceae. All 12 species of fungi that associated with both tree species did so within a 10×40 cm soil volume, suggesting that individual fungal genotypes linked the putatively competing tree hosts.     

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.     

Increasing water‐use efficiency and age‐specific growth responses of old‐growth ponderosa pine trees in the Northern Rockies

We examined radial growth responses of ponderosa pine (Pinus ponderosa var. ponderosa) between 1905–1954 and 1955–2004 to determine if the effects of increased intrinsic water‐use efficiencies (iWUE) caused by elevated atmospheric CO₂ concentrations were age‐specific. We collected 209 cores from five sites in the Northern Rockies and calculated iWUE using carbon isotope data from 1850 to 2004. Standardized radial growth responses were age dependent, with older trees exhibiting significantly higher values than younger trees during the later period at four sites and all sites combined. No significant differences in radial growth existed either for the individual sites or combined site during the earlier period. Increases in iWUE during 1955–2004 were 11% greater than during 1905–1954, and pentadal fluctuations in iWUE were significantly correlated with the radial growth of older trees from 1850 to 2004. Radial growth of younger trees and iWUE were not significantly correlated. Our results suggest that: (1) responses to elevated atmospheric CO₂ in old‐growth ponderosa forests are age‐specific; (2) radial growth increases in older trees coincided with increased iWUE; (3) ponderosa had increased growth rates in their third, fourth, and fifth centuries of life; and (4) age‐specific growth responses during 1955–2004 are unique since at least the mid‐16th century.     

Effect of a non‐host plant Phaseolus vulgaris on larval performance and oviposition of the oriental tobacco budworm Helicoverpa assulta (Lepidoptera: Noctuidae)

The oriental tobacco budworm, Helicoverpa assulta, is a specialist herbivore feeding on a few plants of the Solanaceae family including tobacco. Larval performance and adult oviposition of H. assulta were investigated in a non‐host plant, Phaseolus vulgaris (Fabaceae) in comparison with two solanaceous host plants, Nicotiana tabacum and Datura stramonium. Larvae provided with the P. vulgaris leaf died off at day 15, whereas 50% and 40% of larval populations fed on the leaves of N. tabacum and D. stramonium, respectively, survived at day 15. Larval growth upon feeding showed significant difference between the non‐host plant (P. vulgaris) and the host plants (N. tabacum and D. stramonium), but it was not significantly different between the two host plants. In the no‐choice experiment of oviposition, gravid females laid more eggs in N. tabacum and D. stramonium than in P. vulgaris. When the most likely acceptable host plant, N. tabacum, and the non‐host plant, P. vulgaris, were subjected to the choice experiment of oviposition, H. assulta females preferred to lay eggs in N. tabacum, where eggs were continuously laid during the whole experiment period. However, eggs in P. vulgaris were hardly detected throughout the period. This study showed that the non‐host plant, P. vulgaris, had a negative influence on the larval performance and adult oviposition of H. assulta, implying neonate stage is critical for larval survivorship, and ovipositional preference by the female is highly specialized to host plants. Further investigation is required to identify non‐host factors, which could be applied to the development of alternative pest management strategy against H. assulta.     

Axillary meristems and the development of epicormic buds in wollemi pine (Wollemia nobilis)

Intact trees of Wollemia nobilis Jones, Hill and Allen (Araucariaceae) routinely develop multiple coppice shoots as well as orthotropic epicormic shoots that become replacement or additional leaders. As these are unusual architectural features for the Araucariaceae, an investigation was made of the axillary meristems of the main stem and their role in the production of epicormic and possibly coppice shoots. Leaf axils, excised from the apex to the base of 2-m-high W. nobilis plants (seedling origin, ex situ grown), were examined anatomically. Small, endogenous, undifferentiated (no leaf primordia, no vascular or provascular connections) meristems were found in the axils from near the shoot apex. In the more proximal positions about half the meristems sampled did not differentiate further, but became tangentially elongated to compensate for increases in stem diameter. In the remaining axils the meristems slowly developed into bud primordia, although these buds usually developed few leaf primordia and their apical 'domes' were wide and flat. Associated vascular development was generally restricted to provascular dedifferentiation of the cortical parenchyma, with the procambium usually forming a 'closed loop' that did not extend back to the secondary vascular tissues. Development of the meristems was very uneven with adjacent axils often at widely differing stages of development into buds. The study shows that, unlike most conifers, W. nobilis possesses long-lived meristematic potential in most, if not all, leaf axils. Unlike other araucarias that have been investigated, many of the meristems in the orthotropic main stem will slowly develop into bud primordia beneath the bark in intact plants. It appears likely that this slow but continued development provides a ready source of additional or replacement leaders and thus new branches and leaves.     

Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), a Needle Nematode Parasitizing Pine in Georgia

We describe and illustrate a new needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, (Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with a body length of 5.4-9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end a truncate appearance; long odontostyle (124-165 µm); vulva at 44%-52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required.