Results of the Pine Wilt Disease Surveys in Russia

O.A.Kulinich (1), E.N.Arbuzova (1), N.I.Kozyreva (2), E.S.Mazurin(1)

1) All-Russian Plant Quarantine Center, Bykovo, Moscow oblast, Russia, email: [email protected]; 2)The Center of Parasitology, Institute of Ecology and Evolution of the Russian Academy of Sciences

The wood reserve in Russia (m3 / hectare)

 If the PWN were introduced in Russia, and became widespread there, it is estimated that annual costs could range from 1.3 to 3.7 billion US dollars a year.

 Similar damage will also be true for Europe in the case of PWN spread and lack of control. (Soliman et al., 2012)

Surveys of forests in Russia for the pine wood B. xylophilus, were started early in the 1990s

 In one case the were isolated from dead Korean pine plantations. The number of the nematodes in the wood was 20,000 individuals per g of wood (Kulinich et al., 1994).

Subsequent pathogenicity tests with this isolate conducted in a greenhouse showed that nematode-inoculated 5-year- old Pinus koraiensis and …

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60 Inoculated 40 seedlings 33 40 Control

%, died trees died %, 13 20 0 0 0 Pinus Pinus Pinus koraiensis sylvestris densiflora …larch, Larix olgensis, died after the inoculation.

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80 Inoculated 60 seedlings

40 Control %, died trees 20 0 0 0 0 Larix olgensis Larix dahurica Some other conifer species were resistant to the nematode (Eroshenko & Kruglik, 1998).

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60 Inoculated seedlings 40 Control

%, died trees 20 0 0 0 0 Abies Abies Picea nefrolepis holophylla ajanensis Pest Risk Analysis There are large areas within Russia where the mean July temperature exceeds 20 C. Also, in Russia susceptible host plants and species of longhorn are present. Plant host of Bursaphelenchus xylphilus Vector of Bursaphelenchus xylphilus Pinus sylvestris, sutor, P. sibirica, M. galloprovincialis, P. koraiensis, M. urussovi, P. pumila, M. impluviatus, Picea exelsa, M. nitens, P. obovata, P. ajanensis, M. saltuarius P. koraiensis,

Abies sibirica, A. nephrolepis, A. holophylla, A. sachalinensis,

Larix sibirica L. daurica Large scale surveys were conducted in 2010-2011 where the conifer forest and stored timber were examined for PWN in eleven regions of Russia.

― Bursaphelenchus mucronatus no findings ― Bursaphelenchus mucronatus several findings

 In total, 3,680 samples were analyzed.  B. xylophilus species has not been found in Russia, but the closely related species B.mucronatus was detected.

 The frequency of B. mucronatus occurrence was 11.5% in 2010 and 5.6% in 2011.

 Twenty four isolates of the wood-inhabiting nematode B.mucronatus from 11 Russian regions were extracted and propagated in vitro. Main goal

 What factors affect the pathogenicity of B.xylophilus and B. mucronatus and can B.mucronatus populations induce pine wilt disease, such as the one caused by B. xylophilus?

 The most favourable factors influencing B.xylophilus pathogenicity include: biological characters of the nematode species, and host plant and climate.

 Resent research (2000-2010) in China and South Korea showed that the Pine Wilt Disease of are induced by complex of pathogenic bacteria and the PWN that the bacteria carries (Zhao et al., 2009; Kwon et al., 2010; Wu et al. 2013 and others).

Bacteria associated with wood-inhabiting Bursaphelenchus mucronatus nematodes

Model @ File: 011_F03_1.ab1 Signal G:0 A:0 T:0 C:0 Page 1 of 2 Ð` ƒ ô/ 0/0/0 BIO 1 ?? no 'MTXF' field Spacing: 0 TRACE Lane 0 Points 0 to 0 BioEdit v ersion 7.0.4.1 (2/13/05) Twenty four isolates of the C A AC AC CC T A AAA C T T T CCA C G T GT A CC G T T T CA A C CA A A T A G T T G G G G GT C T T G T C T G G T G GC G G C TG C TG GC T TT AT T G T T G G C G G C TG C TG C TG G G TG AGC C C T A T CA TG G CG AG C G T T TG GG C T TC G GC C TG AG C TAG T AG 10 20 30 40 50 60 70 80 90 100 110 120 130 140 wood-inhabiting nematode

C T T T T C T T T TAA A C C CA T T C C T TA A T A C T G AT T A TA C TG T G G G G AC G A A AG TC C T T G C T T T TA AC T A G AT A G C A A C T T T CA G C AG TG G AT G T C TA G G C T CG C AC A T CG A TG A AG AAC G C T G CG A AC T G C G AT A CG T A A TG C G A AT TBursaphelenchusG C AG G 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 mucronatus were extracted

AT TC AG TG A G T C AT CG A AA T T T T G A AC G C A T A T TG CA C T T C C G GG T T AG T C C TG G G AG T AT G C C TG TA T C AG TG T C C G T AC A T C A A AC T TG G C T T T C T T C C T T C C G TG T AG T C G GT G G AGG AG AT GC C A G ATG T G A AG TG T C T TG C GandG C TG G propagated in vitro to 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 determine the symbiotic

T T T T C G G AC CG AC TG C G AG TC C T T T TA A A T G TA C T G A A C TG T A C T T C T C T T TG C T C G A A A AG C G TG G CG T TG C TG G T TG T G G AG G C TG C T A T T G T A G C A A G T T G GC G AC CG G T T T GT C T G C T G CG G CG T T A A T G G A A G AG T G T TC G AT T C 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 bacteria associated with them.

G CG G T A TG G T TG G C T T C G G C TG A AC A A T G C G C T T A T TG G AT G T T T T T T C T GC T GT G GC G T G A T GG AC C G G TG A A C C A T A G C T C AG T T G C T T G GC T TT T G A AT C G G C T T TG C T G T T GC G A AG T AG A G T G G CG G C T T C G G C TG T C G AG G 600 610 620 630 640 650 660 670 680 690 700 710 720 730Bacterial740 identification was based on sequences 16S rRNA. Results of capillary electrophoresis of the 16S rRNA gene of the bacteria. Twenty species of bacteria belonging to nine families and 9 genera were isolated from the nematodes and identified The bacteria, most frequently encountered, belonged to the genus Pseudomonas (44%). Five species of the genus Pseudomonas were identified Associated bacteria were studied in the dauerlarva stage of B. mucronatus nematodes isolated from the longhorn Monochamus urussovi Fisch. Here, Pseudomonas fluorescens species were extracted from this larval stage of B. mucronatus, as well.  Symbiotic bacteria associated with PWN have been studied in recent years in China, Korea, and Portugal.  However, different researchers extracted different species of pathogenic bacteria which they believe are responsible for the PWD development. Bacillus spp.

Burkholderia Pseudomonas arboris fluorescens Bacteria associated with Bursaphelenchus xylophilus nematodes

 Zhao B., Lin F. Mutualistic  Experiments have symbiosis between shown a high Bursaphelenchus xylophilus and bacteria of the genus degree of Pseudomonas. For Path., 2005, 25, 2205–2214. phytotoxicity of these bacteria:  Kwon Hyeok Ran, Gyung Ja Choi, Yong Ho Choi, Kyoung Soo Jang, P.fluorescens and Nack-Do Sung, Mun Seong Kang, Yilseong Moon, Seung Kyu Lee Burkholderia and Jin-Cheol Kim. Suppression arboris of pine wilt disease by an antibacterial agent, oxolinic acid // Pest Manag. Sci. 2010, 66, 6. P. 634-639. Conclusion

 Thus, Russian B. mucronatus isolates carry bacteria. Perhaps, some populations of this species can vector pathogenic bacteria (for example, Pseudomonas fluorescens) and sometime induce PWD.

It’s assumed that B. mucronatus nematodes and the symbiotic bacterium P. fluorescens can cause death of some Russian pine forests in areas where the mean air temperature in summer months exceeds 250C.

Many people remember the hot summer of 2010. The average monthly temperature in the Centre of European Russia in 2010 was 26.4 ° C in July and 25.5 ° C in August. Following that hot year many dead stands of Pinus sylvestris forests were found.

Сравнительные графики средней температуры воздуха на территории Воронежской области за 2010 г. и за период 2001-2012 гг. t, 0C 30 26,4 25,5

25 22,5 22

17,6 20 18,7 20,8

15,7 14,8 15 14,6 9,5

10 9 7,7 6,3 5 5,3 1,6

0 -0,7 -0,1 -5 -4,1 -3,1 -5,5 -6,7 -5,8 -10

-15 -14,6

-20 Январь Февраль Март Апрель Май Июнь Июль Август Сентябрь Октябрь Ноябрь Декабрь 2001-2012 гг. -5,5 -6,7 -0,1 9 15,7 18,7 22 20,8 14,6 7,7 1,6 -4,1 2010 г. -14,6 -5,8 -0,7 9,5 17,6 22,5 26,4 25,5 14,8 5,3 6,3 -3,1

Russia foresters feel that the death of these trees was caused by drought, but we can not exclude the PWD as having killed the trees. As well, B. mucronatus nematodes and its symbiotic bacteria Pseudomonas fluorescens were extracted from some of the dead trees. Japan Russia