The Pine Wood Nematode: a personal view

The first report of the disease (“pine wilt disease”) associated with the pinewood nematode, goes back to 1905, when Yano reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of he disease was sought, but without success. Because of the large number of insect species that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be one of these. However, in 1971, Kiyohara and Tokushike found a nematode of the genus Bursaphelenchus in infected trees. The nematode found was multiplied on fungal culture, inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii und P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was a named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had suddenly died after the colour of its needles changed to a reddish-brown colour (Dropkin und Foudin, 2 1979). In 1981, B. lignicolus was synonymised by Nickle et al. with B. xylophilus which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides but was recognised by Nickle, in 1970,to belong in the genus Bursaphelenchus. Its common name in the USA was the "pine wood nematode" (PWN. After its detection in Missouri, it became known that B. xylophilus was widespread throughout the USA and Canada. It occurred there on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless susceptible species were stressed eg., by high temperature. This fact was an illuminating piece of evidence that North America could be the homeland of PWN. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the long-horned beetle Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century but in the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. In Japan, there are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN. The occasional transmission by less efficient species of Monochamus or by some of the many other beetle genera in the bark or wood is of little significance. In Europe, M. galloprovincialis and M. sutor transmits the closely related species B. mucronatus. Some speculate that these two insect species are “standing by” and waiting for the arrival of B. xylophilus. In 1982, the nematode was detected and China. It was first found in dead pines near the Zhongshan Monument of Nanjing (CHENG et. al. 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been 3 reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (YANG, 2003). In 1986, the spread of the PWN to Taiwan was discovered and in 1989, the nematode was reported to be present in the Republic of Korea where it had first been detected in Pinus thunbergii and P. densiflora. It was though to have been introduced with packing material from Japan. PWN was advancing. In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the import of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries in 1986 to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognised the potential danger of B. xylophilus for European forests and imposed restrictions on imports into the Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU(92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction

The in vitro cultivation of Bursaphelenchus spp. at the reference laboratory for quarantine pests at Julius Kühn-Institut in Braunschweig

Das Forstquarantänelabor des Julius Kühn-Instituts in Braunschweig (Deutschland) kuratiert eine einzigartige Sammlung lebender Bursaphelenchus-Arten, zudem Dauer­präparate und ITS-RFLP Muster dieser Arten. Die Sammlung wurde von Dr. Helen Braasch gegründet und umfasst derzeit 48 Arten in 305 verschiedenen Isolaten. Diese Isolate wurden über 30 Jahre hinweg weltweit aus verschiedenen Habitaten (Bäumen) und anderen Bezugs­quellen, wie Holzimporten einschließlich Holzverpackungsmaterial, gesammelt. Die Aufzucht der Nema­toden auf sporulierenden und nicht sporulierenden Botrytis cinerea – Kulturen ist anspruchsvoll, arbeitsintensiv und erfordert sowohl Erfahrung als auch Geduld.The Forest quarantine laboratory at Julius Kühn-Institut in Braunschweig (Germany) curates a unique collection of living Bursaphelenchus species, permanent slides and ITS-RFLP profiles. The collection was initiated by Dr. Helen Braasch and currently comprises 48 species in 308 different isolates. These isolates were collected over 30 years across the globe from various habitats and sources, like forest trees and wood imports including wooden packaging material. Cultivation of the nematodes on sporulating and non-sporulating Botrytis cinerea is sophisticated, labor-intensive and requires both, experience and patience

FIGURE 1 in Supplementary characters of Bursaphelenchus lini Braasch, 2004 (Nematoda: Parasitaphelenchidae) and remarks on this nematode

FIGURE 1. Scanning electron micrographs of Bursaphelenchus lini: A. Head region, lateral view; B. Enface view of female head; C. Lateral field; D. Female tail; E. Vulval region; F.­I: Male tail showing papillae (F, G), bursa (H) and protracted spicules (I).Published as part of <i>Braasch, Helen, Brandstetter, Martin & Burgermeister, Wolfgang, 2006, Supplementary characters of Bursaphelenchus lini Braasch, 2004 (Nematoda: Parasitaphelenchidae) and remarks on this nematode, pp. 55-61 in Zootaxa 1141 (1)</i> on page 59, DOI: 10.11646/zootaxa.1141.1.2, <a href="http://zenodo.org/record/10087138">http://zenodo.org/record/10087138</a&gt

FIGURE 2 in Supplementary characters of Bursaphelenchus lini Braasch, 2004 (Nematoda: Parasitaphelenchidae) and remarks on this nematode

FIGURE 2. ITS­RFLP patterns obtained from two individual specimens of Bursaphelenchus lini (left: type A, right: type B). Restriction fragments were obtained by digestion of the amplified rDNA fragment (0) with Rsa I (1), Hae III (2), Msp I (3), Hinf I (4) and Alu I (5). M: DNA size marker (100 bp ladder; Invitrogen Life Technologies).Published as part of <i>Braasch, Helen, Brandstetter, Martin & Burgermeister, Wolfgang, 2006, Supplementary characters of Bursaphelenchus lini Braasch, 2004 (Nematoda: Parasitaphelenchidae) and remarks on this nematode, pp. 55-61 in Zootaxa 1141 (1)</i> on page 60, DOI: 10.11646/zootaxa.1141.1.2, <a href="http://zenodo.org/record/10087138">http://zenodo.org/record/10087138</a&gt

First report of Bursaphelenchus hellenicus Skarmoutsos, Braasch, Michalopoulou 1998 (Nematoda: Aphelenchoididae) from Turkey

Cebeci, H.Huseyin/0000-0002-4569-9527;WOS: 000325500300007A survey was conducted in forest sites of the Mula Regional Forest Directorates for the presence of the pinewood nematode, Bursaphelenchus xylophilus. Wood samples were collected from declining pine and cedar trees. A total of 207 samples were taken, and nematodes were extracted, observed and identified. The pinewood nematode was not detected, but another Bursaphelenchus species was recovered from the wood of a Pinus brutia tree in Een-Karadere location in Fethiye, which strongly resembled the original description of B.hellenicus. It was identified as this species by morphology and ITS-RFLP. Measurements and diagnostic features are presented. This is the first report of B.hellenicus from Turkey.Scientific and Technological Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TOVAG-107O088]This project (TOVAG-107O088) was supported by the Scientific and Technological Council of Turkey (TUBITAK). The authors kindly thank Dr. T. Schroeder, Germany for the confirmation of Bursaphelenchus hellenicus by using the ITS-RFLP technique. We also thank Dr. Besir Yuksel, Ismail Baysal, Metin Serin and Mahir Erdem for their help during the project and the General Directorate of Forestry for their field support. The authors kindly thank Dr. Marc J. Linit for his critical reading of the manuscript. Finally, we would like to thank two anonymous reviewers for their helpful comments on this manuscript

FIGURE 2 in Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group

FIGURE 2. Light photomicrographs of Bursaphelenchus singaporensis sp. n. A, B: Head region; C, D: Female tail; E, F: Male bursa; G, H: Male tail; I: Vulval region. (Scale bars = 10 µm).Published as part of <i>GU, JIANFENG, ZHANG, JIANCHENG, BRAASCH, HELEN & BURGERMEISTER, WOLFGANG, 2005, Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group, pp. 1-12 in Zootaxa 988 (1)</i> on page 6, DOI: 10.11646/zootaxa.988.1.1, <a href="http://zenodo.org/record/10087012">http://zenodo.org/record/10087012</a&gt

FIGURE 3 in Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group

FIGURE 3. Scanning electron microscope (SEM) observations of Bursaphelenchus singaporensis sp. n. A: Head region; B: Female tail; C: Male tail showing caudal papillae, bursa and protruded tips of spicules; D: Female cuticle showing four lateral lines.Published as part of <i>GU, JIANFENG, ZHANG, JIANCHENG, BRAASCH, HELEN & BURGERMEISTER, WOLFGANG, 2005, Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group, pp. 1-12 in Zootaxa 988 (1)</i> on page 7, DOI: 10.11646/zootaxa.988.1.1, <a href="http://zenodo.org/record/10087012">http://zenodo.org/record/10087012</a&gt

FIGURE 4 in Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group

FIGURE 4. ITS­RFLP patterns of Bursaphelenchus singaporensis sp. n. and six morphologically similar species. Each picture shows (from left to right) DNA marker (100 bp ladder, Invitrogen), rDNA amplification product, restriction fragments obtained with Rsa I, Hae III, Msp I, Hinf I, Alu I, and again DNA marker.Published as part of <i>GU, JIANFENG, ZHANG, JIANCHENG, BRAASCH, HELEN & BURGERMEISTER, WOLFGANG, 2005, Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group, pp. 1-12 in Zootaxa 988 (1)</i> on page 9, DOI: 10.11646/zootaxa.988.1.1, <a href="http://zenodo.org/record/10087012">http://zenodo.org/record/10087012</a&gt