J For Res (2011) 16:325–330 DOI 10.1007/s10310-010-0225-6
SHORT COMMUNICATION
An inoculation experiment of Japanese Bursaphelenchus nematodes on Japanese black and red pine, Pinus thunbergii and P. densiflora Natsumi Kanzaki • Takuya Aikawa Noritoshi Maehara • Yu Ichihara
•
Received: 24 June 2010 / Accepted: 13 August 2010 / Published online: 15 September 2010 Ó The Japanese Forest Society and Springer 2010
Abstract The pinewood nematode (PWN) Bursaphelenchus xylophilus is an invasive pathogen that was introduced from North America to Asian countries and Portugal and is devastating native pine forests. Some native European and Asian Bursaphelenchus nematodes also have weak to moderate pathogenicity to native pine species. To evaluate the potential risk of native Bursaphelenchus species, we inoculated ten Japanese Bursaphelenchus species into native pine species (the dominant forest species) in Japan, and evaluated their pathogenicity using mortality and tracheal tissue damage as indices. Inoculation was conducted on August 3, 2007, and the symptoms were observed every 2 weeks until February 1, 2008. None of the inoculated trees, excluding the pathogenic PWN inoculated control, showed external disease symptoms; however, four species [a less pathogenic PWN isolate, B. luxuriosae, Bursaphelenchus sp. NK215 (undescribed), and NK224 (undescribed)] caused tracheal tissue damage in inoculated seedlings and showed weak pathogenicity. Therefore, we conclude that there are some potentially pathogenic native species of nematodes distributed in Japan. Interestingly, two of these weakly pathogenic species, B. luxuriosae and NK215, are not associated with Pinaceae trees, suggesting that nematode pathogenicity may be a pre-adaptive character. More experimental studies under different conditions
N. Kanzaki (&) T. Aikawa Forest Pathology Laboratory, Forestry and Forest Products Research Institute (FFPRI), 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan e-mail:
[email protected] T. Aikawa N. Maehara Y. Ichihara Tohoku Research Center, FFPRI, 92-25 Nabeyashiki, Shimo-Kuriyagawa, Morioka, Iwate 020-0123, Japan
are necessary to accurately evaluate the potential risk of these pathogens. Keywords Bursaphelenchus Japanese black pine Japanese red pine Pathogenicity Potential risk
Introduction Pine wilt disease (PWD), caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus Steiner and Buhrer, is one of the most serious forest pests in East Asia, and the disease has also invaded Portugal (Kishi 1995; Mota et al. 1999). The genus Bursaphelenchus contains two lethal pathogen species: PWN and the red ring nematode B. cocophilus Cobb, which causes palm mortality (Giblin-Davis 1993). Therefore, all members of the genus are treated as important plant quarantine nematodes (Gu et al. 2006a). Although PWN, which is native to North America, is vectored by Asian and European native longhorn beetles, namely, Monochamus alternates Hope and M. galloprovincialis Oliver, respectively (Kobayashi et al. 1984; Sousa et al. 2001), the disease is considered a typical invasive pest with a combination of invasive pathogen versus native susceptible hosts (e.g., Mamiya 2004). Recently, several Bursaphelenchus species native to Asia and Europe have been reported as weak to moderate pine tree pathogens. For example, Braasch (2000) and Kanzaki and Futai (2006) demonstrated that B. mucronatus Mamiya and Enda, a close relative of PWN, shows weak to moderate pathogenicity to European and Asian native pine species Pinus sylvestris L. and P. densiflora Sieb. and Zucc., respectively. Skarmoutsos and MichalopoulosSkarmoutsos (2000) conducted an inoculation test using
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three European Bursaphelenchus species, and concluded that B. sexdentati Ru¨hm and B. leoni Baujard have weak to moderate pathogenicity to P. sylvestris. In the present study, we conducted an inoculation test using ten species of Bursaphelenchus nematodes (PWN plus nine species native to Japan) and two native pine species to evaluate the potential risk of native nematodes to native hosts.
Materials and methods Nematode isolates Ten species of Bursaphelenchus nematodes isolated from various tree and/or insect species served as inoculums. The phylogenetic group, isolation sources, and additional information for each nematode species are shown in Table 1. In advance of the experiment, the nematode species were identified based on morphology and molecular sequences following the phylogenetic classifications suggested by Kanzaki (2008) and Braasch et al. (2009). We tested four xylophilus group species, which are phylogenetically close to PWN species. These included B. doui Braasch, Gu, Burgermeister and Zhang, which is associated with M. subfasciatus Bates and Pinus spp.; B. luxuriosae Kanzaki and Futai, isolated from a species of longhorn beetle (Acalolepta luxuriosa Bates) and which is associated with Araliaceae trees; B. conicaudatus Kanzaki, Tsuda and Futai, which is associated with Psacothea hilaris Pascoe and Moraceae trees; and Bursaphelenchus sp. NK224, isolated from M. grandis Gebler emerged from Abies firma Sieb. and Zucc. We also tested three nonxylophilus group species associated with pine trees, including B. yongensis Gu, Braasch, Burgermeister, Brandstetter and Zhang (eremus group), isolated from Cryphalus fulvus Niijima emerged from dead P. thunbergii Parl.; B. poligraphi Fuchs (sexdentati group); and B. hylobianum Korentchenko (abietinus group), isolated from dead P. thunbergii. The final two species tested were from non-xylophilus groups associated with broad-leaved trees. These included Bursaphelenchus sp. NK 215 (fungivorus group), isolated from Xyleborus seriatus Brandford emerged from a dead log of Quercus crispula Blume, and B. okinawaensis Kanzaki, Maehara, Aikawa and Togashi (okinawaensis group), isolated from M. maruokai Hayashi, which is an endemic species of the southern islands of Japan, usually associated with trees belonging to Lauraceae and Fagaceae (Kanzaki et al. 2008b). The nematodes were reared on gray mould fungus Botrytis cinerea Pers. grown on autoclaved barley grains for 2 weeks, and were isolated from the culture using the
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Baermann funnel technique. Samples were prepared to 100,000 nematodes/ml suspension. Inoculation test and symptom observation The inoculation experiment was conducted on August 3, 2007 in an experimental field at the Forestry and Forest Products Research Institute, Tsukuba, Japan. A 0.05-ml nematode suspension (=5,000 individual mixed-stage nematodes) was inoculated through a drill hole (ø = 4 mm, approximately 1 cm depth) on the main stem of 3-year-old P. thunbergii and P. densiflora seedlings planted directly into the ground. Distilled water was also inoculated as a negative control. The detailed experimental design, i.e. number of inoculated seedlings and tree species, as well as the experimental results are shown in Table 2. The inoculated seedlings were observed for external disease symptoms every 2 weeks until February 1, 2008. Two randomly selected surviving seedlings including their root systems were recovered from each treatment. The roots of these seedlings were washed and immersed in a 0.05% solution of acid fuchsin for several hours; then the shoots from these seedlings were cut 2.0 cm above and below the inoculation site and disease symptoms on the cut surfaces were observed to determine the water flow of the stem tissue. The treatments in which water flow was inhibited in three or four samples (upper ? lower parts from 2 seedlings) were judged as positive for tissue damage.
Results Excluding the positive control, the PWN Ka-4 (pathogenic) isolate which killed 100 and 73% of the P. thunbergii and P. densiflora, respectively, none of the tested seedlings showed any external disease symptoms or mortality (Table 2). However, tissue damage occurred in the P. thunbergii inoculated with the PWN OKD-1 (less pathogenic) isolate, B. luxuriosae, Bursaphelenchus sp. NK215, and Bursaphelenchus sp. NK224. The intensity of water flow inhibition caused by the latter three species seemed similar to each other and to that caused by the less pathogenic PWN isolate (Fig. 1).
Discussion Among the nine native species of nematodes tested, B. luxuriosae, Bursaphelenchus sp. NK215, and Bursaphelenchus sp. NK224 caused tissue damage on P. thunbergii at an intensity similar to the less pathogenic PWN,
Okayama, Okayama 1984
Kyoto, Kyoto
Izu, Sizuoka
Happo, Akita
Gose, Nara
Ishigaki, Okinawa
Otsuki, Iwate
Hiki, Kagoshima
Asahi, Yamagata
Hachioji, Tokyo
B. xylophilus OKD-1 (less pathogenic)
B. conicaudatus
B. doui
B. hylobianum
B. luxuriosae
B. okinawaensis
B. poligraphi
B. yongensis
Bursaphelenchus sp. NK215
Bursaphelenchus sp. NK224
2006
2006
2006
2006
2006
2001
2006
1995
1999
1994
Kasama, Ibaraki
Bursaphelenchus xylophilus Ka-4 (pathogenic)
Aralia elata
P. thunbergii
P. densiflora
Ficus carica
P. thunbergii
Pinus densiflora
xylophilus
fungivorus
eremus
sexdentati
Cryphalus fulvus
Unknown
M. maruokai
Abies firma
M. grandis
–
–
P. massoniana (China)
Picea spp. (EU)
–
Kanzaki et al. (2000), Kanzaki and Futai (2001, 2002)
Summarised in Ryss et al. (2005)
Summarised in Ryss et al. (2005)
References
–
–
–
Polygraphus poligraphus, Hylurgops palliatus (EU)
–
–
–
–
Gu et al. (2006b)
Summarised in Ryss et al. (2005)
Kanzaki et al. (2008b)
Kanzaki and Futai (2003)
Summarised in Ryss et al. (2005)
M. subfasciatus (Japan) Braasch et al. (2005), Kanzaki et al. (2008a)
–
–
Previously reported carrier insect (country)
Pinus spp. (Russia, EU) Hylobius albosparsus (Russia, EU)
‘‘Coniferous packing material’’ (East Asia)
Morus bombycis, M. australis (Japan)
–
–
Previously reported host plant (country)
Acalolepta luxuriosa –
Unknown
unknown
Psacothea hilaris
Monochamus spp.
Monochamus spp.
Source carrier insect
Quercus crispula Xyleborus seriatus
P. thunbergii
P. thunbergii
okinawaensis Unknown
xylophilus
abietinus
xylophilus
xylophilus
xylophilus
xylophilus
Isolation Intra-generic Source host year group plant
Locality (city, prefecture)
Species (isolate) name
Table 1 Origin, source, and isolation information for the nematode isolates
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Table 2 Experimental design and the inoculation experiment results Nematode species (isolate)
Pinus thunbergii
P. densiflora No. of dead seedlings/number of inoculated seedlings
Tracheal tissue damagea
No. of dead seedlings/number of inoculated seedlings
Tracheal tissue damagea
Bursaphelenchus xylophilus Ka-4
30/30
Not examined
22/30
Not examined
B. xylophilus OKD-1
0/30
?
0/30
-
B. conicaudatus B. doui
0/30 0/30
-
0/30 0/30
-
B. hylobianum
0/30
-
0/30
-
B. luxuriosae
0/30
?
0/30
-
B. okinawaensis
0/30
-
0/30
-
B. poligraphi
0/30
-
0/30
-
B. yongensis
0/8b
-
N/Ab
N/Ab
b
N/Ab N/Ab
Bursaphelenchus sp. NK215
0/30
?
N/A
Bursaphelenchus sp. NK224
0/15b
?
N/Ab
a
?, tissue damage (water flow inhibition) observed; -, tissue damage not observed
b
Inoculum nematodes were insufficient for the experiment because of their culture condition
Fig. 1 Cut surface of Pinus thunbergii seedlings showing water flow inhibition. a Control (distilled water); b Bursaphelenchus xylophilus isolate OKD-1 (less pathogenic); c B. luxuriosae; d Bursaphelenchus sp. NK215; e Bursaphelenchus sp. NK224
which sometimes kills pine trees (e.g., Takeuchi and Futai 2007). Thus, these three species could be potentially pathogenic for native pines in Japan. However, B. luxuriosae and Bursaphelenchus sp. NK215 were isolated from A. luxuriosa and X. seriatus, respectively, and neither of these beetle species are associated with pine trees, although X. seriatus is associated with a wide range of coniferous and broad-leaved trees (Hayashi et al. 1984; Ohbayashi and Niisato 2007). In contrast, M. grandis, the carrier of
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Bursaphelenchus sp. NK224, is associated with a wide range of Abies spp. and Pinaceae trees, including native Japanese pine (Ohbayashi and Niisato 2007). Therefore, we conclude that only Bursaphelenchus sp. NK224 should be regarded as a potential risk to native pine trees. Our results also showed no clear correlation between pathogenicity and habitat (host) preference or nematode phylogenetic group; that is, the two xylophilus group species B. doui and B. conicaudatus and two Pinus-associated
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species B. yongensis and B. poligraphi, which is a close relative of the weak/moderate pathogen B. sexdentati (Lange et al. 2007), did not show clear pathogenicity. Braasch et al. (1998) and Braasch (2000) demonstrated that B. mucronatus shows strong pathogenicity, causing 100% mortality in P. sylvestris under high temperature and/ or drought conditions, and Skarmoutsos and Michalopoulos-Skarmoutsos (2000) demonstrated that the pathogenicity of B. sexdentati and B. leoni (leoni group: phylogenetic position unknown) is enhanced by high temperature and/or drought in a greenhouse experiment. Kanzaki and Futai (2006) found that shading enhanced the pathogenicity of B. mucronatus to P. densiflora in a field experiment with potted seedlings. For a more accurate evaluation of the potential risk of native Japanese nematodes, experiments that consider climate change by, for example, simulating drought or high temperature conditions will be necessary. Several studies have reported the isolation of Bursaphelenchus nematodes from declining pine species in Europe (e.g., Penas et al. 2004; Polomski et al. 2006). We believe that these kinds of risk evaluations in all regions in which native Bursaphelenchus species and related nematodes occur are necessary to develop worldwide risk management. Pinus thunbergii is more susceptible to PWD than is P. densiflora (e.g., Kishi 1995). In the present study, clear tissue damage (water flow inhibition) occurred only in P. thunbergii (Table 2), suggesting that the pathogenicity/ tolerance mechanism of the native nematode–native pine system is similar to that of the PWD system. However, the pathogenic mechanisms of PWD and the origin of PWN pathogenicity have not been fully clarified. If the pathogenic mechanism of these three weak pathogens is the same as that of PWN, the pathogenicity is probably derived from some kind of tolerance of these nematodes to chemical substances in living trees, e.g., pinene or terpenoids, and could be regarded as a pre-adaptive character. Further pathological studies on the PWN system and other species and physiological studies on several different Bursaphelenchus nematodes are necessary. Acknowledgments We are grateful to Dr. Hayato Masuya of FFPRI for his comments on the experimental design and for technical assistance in sampling. This study was supported by the Global Environment Research Fund (D-0801; F-081), and the Projective Fund of ‘‘Development of Mitigation and Adaptation Techniques to Global Warming in the Sectors of Agriculture, Forestry, and Fisheries’’ by the Ministry of Agriculture, Forestry and Fishery, Japan.
References Braasch H (2000) Influence of temperature and water supply on mortality of 3-year-old pines inoculated with Bursaphelenchus xylophilus and B. mucronatus. Nachr Dtsch Pflanzenschutzd 52:244–249
329 Braasch H, Caroppo S, Ambrosioni L, Michalopoulos H, Skarmoutsos G, Tomiczek CH (1998) Pathogenicity of various Bursaphelenchus species and implication to European forests. In: Futai K, Togashi K, Ikeda T (eds) Sustainability of pine forests in relation to pine wilt and decline. Shokado, Tokyo, pp 14–22 Braasch H, Gu J, Burgermeister W, Zhang J (2005) Bursaphelenchus doui sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Taiwan and South Korea—a new species of the xylophilus group. Russ J Nematol 12:19–27 Braasch H, Burgermeister W, Gu J (2009) Revised intra-generic grouping of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae). J Nematode Morphol Syst 12:65–88 Giblin-Davis RM (1993) Interactions of nematodes with insects. In: Wajid M (ed) Nematode interactions. Chapman & Hall, London, pp 302–344 Gu J, Braasch H, Burgermeister W, Zhang J (2006a) Records of Bursaphelenchus spp. intercepted in imported packaging wood at Ningbo, China. For Pathol 36:323–333 Gu J, Braasch H, Burgermeister W, Brandstetter M, Zhang J (2006b) Description of Bursaphelenchus yongensis sp. n. (Nematoda: Parasitaphelenchidae) isolated from Pinus massoniana in China. Russ J Nematol 14:91–99 Hayashi M, Morimoto K, Kimoto S (1984) The Coleoptera of Japan in color (IV). Hoikusha, Tokyo (in Japanese) Kanzaki N (2008) Taxonomy and systematics of the nematode genus Bursaphelenchus (Nematoda: Parasitaphelenchidae). In: Zhao BG, Futai K, Sutherland J, Takeuchi Y (eds) Pine wilt disease. Springer, Japan, pp 44–66 Kanzaki N, Futai K (2001) Life history of Bursaphelenchus conicaudatus (Nematoda: Apehelenchoididae) in relation to the yellow-spotted longicorn beetle, Psachothea hilaris (Coleoptera: Cerambycidae). Nematology 3:473–479 Kanzaki N, Futai K (2002) Phylogenetic analysis of the phoretic association between Bursaphelenchus conicaudatus (Nematoda: Aphelenchoididae) and Psacothea hilaris (Coleoptera: Cerambycidae). Nematology 4:759–771 Kanzaki N, Futai K (2003) Description and phylogeny of Bursaphelenchus luxuriosae n. sp. (Nematoda: Aphelenchoididae) isolated from Acalolepta luxuriosa (Coleoptera: Cerambycidae). Nematology 5:565–572 Kanzaki N, Futai K (2006) Is Bursaphelenchus mucronatus a weak pathogen to the Japanese red pine? Nematology 8:485–489 Kanzaki N, Tsuda K, Futai K (2000) Description of Bursaphelenchus conicaudatus n. sp. (Nematoda: Aphelenchoididae), isolated from the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae) and fig trees, Ficus carica. Nematology 2:165–168 Kanzaki N, Aikawa T, Maehara N, Matsumoto K (2008a) Bursaphelenchus doui Braasch, Gu, Burgermeister & Zhang, 2005 (Aphelenchida: Parasitaphelenchidae), an associate of Monochamus subfasciatus Bates (Coleoptera: Cerambycidae) and Pinus densiflora Sieb. & Zucc. Nematology 10:69–78 Kanzaki N, Maehara N, Aikawa T, Togashi K (2008b) First report of parthenogenesis in the genus Bursaphelenchus Fuchs, 1937: a description of Bursaphelenchus okinawaensis sp. nov. isolated from Monochamus maruokai (Coleoptera: Cerambycidae). Zool Sci 25:861–873 Kishi Y (1995) The pine wood nematode and the Japanese pine sawyer. Thomas, Tokyo, p 302 Kobayashi F, Yamane A, Ikeda T (1984) The Japanese pine sawyer beetle as the vector of pine wilt disease. Annu Rev Entomol 29:115–135 Lange C, Burgermeister W, Metge K, Braasch H (2007) Phylogenetic analysis of isolates of the Bursaphelenchus sexdentati group using ribosomal intergenic transcribed spacer DNA sequences. J Nematode Morphol Syst 9:95–109
123
330 Mamiya Y (2004) Pine wilt disease in Japan. In: Mota MM, Vieira P (eds) The pine wood nematode, Bursaphelenchus xylophilus. Nematology monographs and perspectives, vol 1. Brill, Leiden, pp 9–20 Mota MM, Braasch H, Bravo MA, Penas AC, Burgermeister W, Metge K, Sousa E (1999) First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1:727–734 Ohbayashi N, Niisato T (2007) Longicorn beetles of Japan. Tokai University Press, Tokyo (in Japanese) Penas AC, Correia P, Bravo MA, Mota MM, Tenreiro R (2004) Species of Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) associated with maritime pine in Portugal. Nematology 6:437–453 Polomski J, Scho¨nfeld U, Braasch H, Dobbertin M, Burgermeister W, Rigling D (2006) Occurrence of Bursaphelenchus species in declining Pinus sylvestris in a dry Alpine valley in Switzerland. For Pathol 36:110–118
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J For Res (2011) 16:325–330 Ryss A, Vieira P, Mota MM, Kulinich O (2005) A synopsis of the genus Bursaphelenchus Fuchs, 1937 (Aphelenchida: Parasitaphelenchidae) with keys to species. Nematology 7:393–458 Skarmoutsos G, Michalopoulos-Skarmoutsos H (2000) Pathogenicity of Bursaphelenchus sexdentati, Bursaphelenchus leoni and Bursaphelenchus hellenicus on European pine seedlings. For Pathol 30:149–156 Sousa E, Bravo M, Pires J, Naves P, Penas AC, Bonifa´cio L, Mota MM (2001) Bursaphelenchus xylophilus (Nematoda; Aphelenchoididae) associated with Monochamus galloprovincialis (Coleoptera; Cerambycidae) in Portugal. Nematology 3:89–91 Takeuchi Y, Futai K (2007) A virulent isolate of the pinewood nematode Bursaphelenchus xylophilus, survives 7 months in asymptomatic host seedlings. For Pathol 37:289–291
