Rina Sriwati

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom Tricholoma matsutake

AbstactThe fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ’shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.

Bursaphelenchus eproctatus n. sp. (Nematoda: Parasitaphelenchidae) isolated from dead Japanese black pine, Pinus thunbergii Pars.

Bursaphelenchus eproctatus n. sp. is described and figured from a dead Japanese black pine, Pinus thunbergii. The new species has a body length of 639 (580-714) μm in the female and 545 (550-595) μm in the male, ratio a = 35.9 (32.6-41.9) in the female and 33.8 (31.5-35.1) in the male, and c and c′ ratios = 14.2 (13.3-15.0) and 2.7 (2.3-3.0), respectively, in the male. A functional rectum and anus are absent, the intestine ending as a blind sac in the female posterior region. The female stylet = 18.8-21.8 μm long and the male stylet = 14.9-19.8 μm, the median bulb is elongate-oval and there are three lines in the lateral field. The spicules are of medium size, 19.8 (18.8-20.8) μm long, and arcuate with a terminal cucullus; there are two pairs of male caudal papillae and the female tail is tapered with a rounded terminus and a clearly annulated dorsal surface near the tail tip. Based upon spicule morphology the new species belongs to the B. piniperdae-group sensu Ryss and is close to B. hunanensis, B. minutus and B. lini. However, the new species is distinguished from these three species by a number of morphological features and morphometric values.

Cohabitation of the pine wood nematode, Bursaphelenchus xylophilus , and fungal species in pine trees inoculated with B. xylophilus

The relationship between the pine wood nematode (PWN), Bursaphelenchus xylophilus, and fungi cohabiting in 15-year-old Japanese black pine, Pinus thunbergii, was examined at intervals of 2 months over a year following inoculation with PWN. The population of PWN in the trees was large in August, but decreased slightly in December then increased again in February. Eighteen species of fungi were isolated from wood samples of the pine trees examined. Among them were Phialophora repens, Sphaeropsis sapinea, Pestalotiopsis spp. and Rhizoctonia sp., which were detected most frequently every season. All of these dominant fungi had positive effects on increasing the nematode population except for Rhizoctonia sp., on which PWN propagated less. Under laboratory conditions, 18 species of fungi isolated from pine trees and Botrytis cinerea cultured on potato dextrose agar served as food for PWN, and PWN population build up was compared at 20°C. PWN increased significantly on Pestalotiopsis sp. 1, Pestalotiopsis sp. 2, Sphaeropsis sapinea, Phialophora repens and B. cinerea from 10-15 days after inoculation. From the viewpoint of food quality and their cohabiting ability, we concluded that the species of fungi that were dominant in the pine trees, except for Rhizoctonia sp., had a compatible relationship with PWN, whilst Rhizoctonia sp. and Penicillium spp. were neutral, and Trichoderma spp. had an incompatible relationship with PWN.

Aphelenchoidid nematodes associated with two dominant Ficus species in Aceh, Indonesia

Aphelenchoidid nematodes associated with the syconia of two dominant fig species, Ficus hispida and F. racemosa , were surveyed in Banda Aceh, Indonesia. Nematodes were isolated from sycones and pollinating wasps of these two fig species from four localities in the area, and identified based on the molecular sequences of two genetic loci, D2-D3 expansion segments of large subunit ribosomal RNA (D2-D3 LSU) and mitochondrial cytochrome oxidase subunit I (mtCOI). Molecular sequences of D2-D3 LSU and mtCOI were successfully determined for 44 and 19 individual nematodes, respectively, and these sequences were separated into four clades, i.e. , types A-D of D2-D3 LSU and types I-IV of mtCOI. Phylogenetic analysis of the DNA sequences deposited in the GenBank database showed that the DNA sequences corresponded to three species, namely, Martininema baculum (type B/II), Ficophagus fleckeri (types A/I, D/IV) and F . cf. centerae (type C/III). Within these species, F. fleckeri was separated into two clades as suggested in previous studies and thus it may possibly reflect the existence of two different taxa, F. fleckeri and a cryptic species. The Indonesian F . cf. centerae was monophyletic with, but clearly separated from, the Chinese population of F. centerae and thus the Indonesian population is potentially an undescribed species. Overall, the species composition of fig-associated aphelenchoidids in the Aceh region seemed intermediate between continental Chinese and Australian species. However, further material collections followed by detailed morphological analyses are necessary to characterise or describe these fig-associated aphelenchoidids in Indonesia.

First record of Hammerschmidtiella diesingi (Hammerschmidt, 1838) (Oxyuridomorpha: Thelastomatidae) parasite of Periplaneta americana (Linnaeus, 1758) (Blattodea: Blattidae) in Japan, morphological and molecular characterization

Nematodes, belonging to the family Thelastomatidae, are parasites of saprophytic terrestrial arthropods, mainly cockroaches. American cockroach Periplaneta americana (Linnaeus, 1758) has high environmental adaptability, and has been spread worldwide by human activity. There are several reports of thelastomatid parasitic nematodes of P americana in the world. Here, the thelastomatid nematode Hammerschmidtiella diesingi (Hammerschmidt, 1838) (Oxyuridomorpha: Thelastomatidae), isolated from P americana is recorded for the first time in Japan. Through morphometrics, DIC and SEM observations, we confirmed that specimens of the present study agree with previous records of H. diesingi. DNA sequencing of the partial D2/D3 LSU expansion segment of the 28S ribosomal RNA gene revealed that H. diesingi from Japan matches with H. diesingi from other countries. Our contributions are very useful and fundamental for further analysis of the cockroach and parasite relations.

Nematode Fauna and Fungal Flora in Infected Pine Trees

In Japan the pine wood nematode (PWN) is transmitted mainly by the Japanese pine sawyer, Monochamus alternatus, from wilt-killed to healthy pine trees (Mamiya and Enda 1972; Morimoto and Iwasaki 1972). The adult beetles of M. alternatus carry a great number of PWNs in their tracheae when they emerge from PWN-killed pines in early summer. Newly emerging adults fly to healthy trees and feed on the bark of young twigs for maturation of their reproductive organs (maturation feeding). At that time, the PWNs on the vector beetles are transmitted to healthy trees and invade them through the feeding wounds made by the beetles. A small number of PWNs disperse widely in the infected trees and causes cessation of oleoresin flow. Thereafter, PWNs propagate dramatically and the trees show wilting symptoms, releasing volatiles such as ethanol, and terpenes (reviewed by Kishi 1995). Mature beetles are attracted to these wilting trees and oviposit in them. The eggs hatch within a week and the larvae feed on the inner bark and outermost sapwood, and then in autumn bore into the sapwood to form pupal chambers (PCs). The number of PWNs reaches its maximum from autumn to winter, and then decreases gradually (Mamiya et al. 1973; Fukushige and Futai 1987). The PC of M. alternatus beetles is one of the most important places for PWNs, because as Mamiya (1972) reported numerous PWNs aggregated around the PCs of M. alternatus in wilt-killed pine trees and that the beetles emerging in the subsequent year harbored many nematodes on their bodies.

The Relationship Between the Pinewood Nematode (PWN) and Fungi Cohabiting in aine Trees Inoculated with the PWN

The relationship between the pinewood nematode (PWN) and fungi cohabiting with the nematodes in 15-year-old Japanese black pine (Pinus thunbergii) was examined bimonthly over a year after inoculation with PWN. The population of PWN in the trees was high in August, but slightly decrease in December then increased again in February. From wood samples of the pine trees examined, 18 species of fungi have been isolated. Among the 18 fungi detected, Phialophora repens, Sphaeropsis sapinea, Pestalotiopsis sp., Rhizoctonia sp. were the most frequently isolated in every season. All of these fungi had positive effects on the increase of nematode population, though the population of PWN on Rhizoctonia sp. was less than those on the other three dominant fungi. Under laboratory conditions, 19 species of fungi cultured on potato dextrose agar (PDA) served for PWN as food source, and the PWN’s population built up on each fungus was compared at 20 ˆC. PWN dramatically increased on Pestalotiopsis sp. 1, Pestalotiopsis sp. 2, Sphaeropsis sapinea, Phialophora repens, and Botrytis cinerea (control), from 10 to 15 days after inoculation. From the point of view in terms of the food quality and their cohabitating ability we conclude that the species of fungi that are dominant in pine trees, except Rhizoctonia sp., have a compatible relationship with the PWN, while Rhizoctonia sp. and Penicillium sp. proved to be neutral, and Trichoderma sp. an incompatible relationship with PWN.