Kazuyoshi Futai

A PCR primer set for determination of phylogenetic relationships of Bursaphelenchus species within the xylophilus group

The phylogenetic relationships of Bursaphelenchus conicaudatus with B. abruptus, B. fraudulentus, B. mucronatus and B. xylophilus species were analysed based on the DNA base sequences of 18S, 5.8S, ITS1 and ITS2 of rDNA and the partial code of mitochondrial cytochrome oxidase subunit I (COI) gene. The monophyly of B. abruptus and the other species in the xylophilus group was not supported, and B. abruptus could be excluded from the xylophilus group. The other species, i.e ., B. conicaudatus, B. fraudulentus, B. mucronatus and B. xylophilus were assumed to be monophyletic. The ancestor of these four species is assumed to have originated in the eastern part of the Eurasian continent as a free-living nematode inhabiting broad-leaved trees. First, B. conicaudatus branched from the ancestor in eastern Asia. Then B. fraudulentus and B. mucronatus separated from the ancestor. B. mucronatus changed its host from broad-leaved trees to conifers and spread throughout the coniferous forest over the Eurasian continent and North America. B. xylophilus might originate from a population of B. mucronatus remaining in North America after B. mucronatus had diversified.

Mycorrhizae : sustainable agriculture and forestry

Preface 1. Mycorrhizae: An overview Z.A. Siddiqui, J. Pichtel 2. The molecular components of nutrient exchange inarbuscular mycorrhizal interactions J.H. Ruairidh, S-Y. Yang, C. Gutjahr, U. Paszkowski 3. Arbuscular mycorrhizal fungi as potential bioprotectants against plant pathogens M.S. Akthar, Z.A. Siddiqui 4. Arbuscular Mycorrhizae and alleviation of soil stresses P. Giasson, A. Karam, A. Jaouich 5. Arbuscular mycorrhizal fungi communities in major intensive North American grain productions M.S. Beauregard, C. Hamel, m. St-Arnaud 6. Arbuscular mycorrhizae: A dyanamic microsymbiont for sustainable agriculture J. Panwar, R.S. Yadav, B.K. Yadav, J.C. Tarafdar 7. Indirect contributions of AM fungi and soil aggregation to plant growth and protection K.A. Nichols 8. Arbuscular mycorrhizae and their role in plant restoration in native ecosystems K. Jayachandran, J. Fisher 9. Effects of interactions of arbuscular mycorrhizal fungi and beneficial saprophytic mycoflora on plant growth and disease protection M.G.B. Saldajeno, W.A. Chandanie, M. Kubota, M. Hyakumachi 10. The mycorrhizosphere effect: A multitrophic interaction complex improves mycorrhizal symbiosis and plant growth R. Duponnois, A. Galiana, Y. Prin 11. Ectomycorrhizae and their importance in forest ecosystems K. Futai, T. Taniguchi, R. Kataoka 12. Ectomycorrhizal associations function to maintain tropical monodominance K.L. McGuire 13. The use of mycorrhizal biotechnology in restoration of disturbed ecosystem A.M. Quoreshi 14. In vitro mycorrhization of micropropagated plants: Studies on Castanea sativa Mill A. Martins 15. Effective and flexible methods for visualizing and quantifying endorhizal fungi S.G.W. Kaminskyj Subject index

Pine Wood Nematode, Bursaphelenchus xylophilus

After devastating vast areas of pine forests in Asian countries, the pine wilt disease spread into European forests in 1999 and is causing worldwide concern. This disease involves very complicated interactions between a pathogenic nematode, its vector beetle, host pine species, and fungi in dead hosts. Pathogenicity of the pine wood nematode is determined not only by its physical and chemical traits but also by its behavioral traits. Most life history traits of the pine wood nematode, such as its phoretic relationship with vector beetles, seem to be more effective in virulent than in avirulent isolates or species. As the pathogenicity determinants, secreted enzymes, and surface coat proteins are very important, they have therefore been studied intensively. The mechanism of quick death of a large pine tree as a result of infection by a tiny nematode could be ascribed to the dysfunction of the water-conducting system caused by the death of parenchyma cells, which must have originally evolved as an inherent resistant system.

Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate.

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.

MODELING THE SPREAD OF PINE WILT DISEASE CAUSED BY NEMATODES WITH PINE SAWYERS AS VECTOR

An epidemic of pine wilt disease has been spreading in wide areas of Japan for nearly a century. The disease is caused by the pinewood nematode, Bursaphelenchus xylophilus, with the pine sawyer, Monochamus alternates, as vector. The spread of disease is facilitated by an obligatory mutualism between the nematode and the pine sawyer: the pine sawyer helps the nematode transmit to a new host tree, while the nematode supplies the pine sawyer with newly killed trees on which to lay eggs. We present a mathematical model to describe the host-vector interaction between pines and pine sawyers carrying nematodes, on the basis of detailed data on the population dynamics of pine sawyers and the incidence of pine wilt disease at a study site located on the northwest coast of Japan. We used the model to simulate the dynamics of the disease and predict how the epidemic could be controlled by eradication of the pine sawyer. The main results are as follows: (1) There is a minimum pine density below which the disease always fails in invasion. However, even if the pine density exceeds this minimum, the disease fails in invasion due to the Allee effect when the density of pine sawyers is very low. (2) The minimum pine density increases disproportionately with increase in the eradication rate. (3) The probability that a healthy tree will escape from infection until the epidemic dies out decreases sharply with increase in the initial pine density or the initial density of pine sawyers.

Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry

Since it was first introduced into Asia from North America in the early 20th century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine’s proteins. These proteins could have been acquired by host–parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.

A nested PCR-based method for detecting the pine wood nematode, Bursaphelenchus xylophilus , from pine wood

Several methods were examined for extracting DNA from the woody tissues of Japanese black pine, including the general CTAB method which proved to be the most successful. To detect the pine wood nematode, the causal agent of pine wilt disease (PWD) in pine wood tissues, PWN-specific PCR was done using a DNA mixture obtained from 80 mg of pine wood tissues and a single pine wood nematode as the DNA template. The results showed that nested PCR could amplify the ITS region specific to the PWN, even though the amount of nematode DNA was extremely small. Also, the PWN could be detected in diseased tissues of several Pinus spp. which had been inoculated with nematodes. The method described here can also be used as a simple and quick detection method for a wide range of wood-inhabiting pathogens.

Role of asymptomatic carrier trees in epidemic spread of pine wilt disease

To determine why pine wilt disease caused by the pinewood nematode (Bursaphelenchus xylophilus) recurs in the same pine stand even after thorough eradication of dead pine trees, the amount of oleoresin exudation from artificial wounds was measured from 72 Pinus koraiensis trees, highly susceptible to this disease, for 4 years. The amount of exuded oleoresin was rated from 0 to 4. All values obtained for each tree were summed at each measurement; thereby a cumulative curve was drawn to monitor the physiological condition of each test tree. Cumulative curves suggest that some pine trees that died had already been infected in the previous year or earlier and then had survived without any visible symptoms. If cessation of oleoresin exudation delays, and overlaps with activity of Monochamus alternatus, the vector beetle of pinewood nematodes, in the following season, such trees can be referred to as “latent carriers” or “asymptomatic carriers”. They could play a significant role as attractants for M. alternatus that could then transmit B. xylophilus to neighboring trees. Behavior of M. alternatus caged with several pine seedlings, only one of which acted as an asymptomatic carrier of B. xylophilus, confirmed this idea. The presence of asymptomatic pine trees, which harbored B. xylophilus nematodes or had reduced annual elongation, near stumps of newly dead trees in the study stand also substantiated this hypothesis.

Description of Bursaphelenchus conicaudatus n. sp. (Nematoda: Aphelenchoididae), isolated from the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae) and fig trees, Ficus carica.

Bursaphelenchus conicaudatus n. sp. is described and figured. Specimens were collected from a 3-week-old culture on Botrytis cinerea. The new species is characterised by the roundish trapezoid bursa of males and tapered and mucronate tail of females. The new species is closely related to B. xylophilus, B. mucronatus and B. fraudulentus in the shapes of spicule and vulval flap, but was easily distinguished from these three species by the shapes of the bursa and female tail. Bursaphelenchus conicaudatus n. sp. est decrit et illustre. Des specimens ont ete collectes a partir d’une culture de 3 semaines sur Botrytis cinerea. La nouvelle espece est caracterisee par une bourse trapezoidate et arrondie chez le mâle et par l’extremite mucronee de la queue chez la femelle. La nouvelle espece est tres proche de B. xylophilus, B. mucronatus et B. fraudulentus par la forme des spicules et du volet vulvaire mais elle a ete facilement differenciee de ces trois especes par la forme de la bourse du mâle et de la queue de la femelle.

Biocontrol of Meloidogyne incognita on tomato using antagonistic fungi, plant-growth-promoting rhizobacteria and cattle manure

BACKGROUND Biocontrol achieved by a single biocontrol agent is generally inconsistent under field conditions. The aim of the present study was to increase the competitiveness and efficacy of biocontrol agents by using them together with cattle manure. RESULTS The effects of antagonistic fungi [Aspergillus niger v. Teigh., Paecilomyces lilacinus (Thom) Samson and Penicillium chrysogenum Thom] and plant-growth-promoting rhizobacteria (PGPR) [Azotobacter chroococcum Beijer., Bacillus subtilis (Ehrenberg) Cohn and Pseudomonas putida (Trev.) Mig.] were assessed with cattle manure on the growth of tomato and on the reproduction of Meloidogyne incognita (Kof. & White) Chitwood. Application of antagonistic fungi and PGPR alone and in combination with cattle manure resulted in a significant increase in the growth of nematode-inoculated plants. The highest increase (79%) in the growth of nematode-inoculated plants was observed when P. putida was used with cattle manure, followed by use of P. lilacinus plus cattle manure. Paecilomyces lilacinus resulted in a high reduction in galling and nematode multiplication, followed by P. putida, B. subtilis, A. niger, A. chroococcum and P. chrysogenum. The combined use of P. lilacinus with cattle manure resulted in a maximum reduction in galling and nematode multiplication. CONCLUSION Application of P. lilacinus or P. putida with cattle manure was useful to achieve greater biocontrol of M. incognita on tomato.