Journal of Nematology Volume 43 September–December 2011 Numbers 3–4
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JOURNAL OF NEMATOLOGY VOLUME 43 SEPTEMBER–DECEMBER 2011 NUMBERS 3–4 Journal of Nematology 43(3–4):129–134. 2011. Ó The Society of Nematologists 2011. The role of wood-inhabiting bacteria in pine wilt disease BO GUANG ZHAO,JIAN TAO,YUN WEI JU,PENG KAI WANG,JIAN LING YE Abstract: The pathogenicity of the pine wood nematode (PWN), Bursaphelenchus xylophilus together with the bacteria isolated from black pine (Pinus thunbergii) bark inoculated to axenic black pine seedlings, significantly exceeded that of the axenic PWNs alone, demonstrating that the bacteria play an important role in pine wilt disease. Inoculation of seedlings with bacteria-free culture filtrates of the seven isolates from the dead seedlings from the above experiment showed that all isolate filtrates killed the seedlings within 8 days. Identification of the bacteria using 16S rDNA sequencing showed that the isolates belonged to strains By253Ydz-fq, S209, 210-50 and 210-50 in Bacillus and the DN1.1 strain of Stenotrophomonas maltophilia, respectively. Completing Koch’s postulates using the seven bacterial isolates to inoculate pine seedlings showed that all the seedlings that received aseptic PWNs mixed with the seven bacterial isolates died within 18 days post inoculation, while those inoculated with ‘wild’ PWNs died 16 days post inoculation. No disease symptoms developed on seedlings that received sterile water or aseptic PWNs. The horizontal transfer of the pathogenic bacteria may explain differences in bacterial species carried by PWN in different geographic areas. Key words: Bursaphelenchus xylophilus, ecology, forest bacteria, pine wilt disease, Pinus thunbergii. Pine wilt disease (PWD) causes serious damage to pine evolve as a pathogen on living pine hosts. The perpet- forests in Japan, China (Mamiya, 1983; Cao and Han, uation of symbiosis through host generations relies on 2001) and Western Europe (Mota and Vieira, 2008). symbiont transmission (Bright and Bulgheresi, 2010). Earlier studies indicated that the disease is caused solely Microbial mutualism is the basis for the evolution of the by the pinewood nematode (PWN), Bursaphelenchus xy- eukaryotic cell and allows organisms to exploit inac- lophilus (Mamiya, 1983; Mayer, 1988). Several studies cessible niches (Chaston and Goodrich, 2010). To un- have described bacteria associated with PWN (Oku derstand this evolutionary process, it is important to et al., 1980; Higgins et al., 1999); and bacteria were ob- know where and how PWN acquires its associated path- served, by using scanning electron microscopy, on the ogenic bacteria. Since adult PWNs, and their offspring surface of the nematodes (Zhao et al., 2000). Axenic live in diseased trees, the offspring might acquire bac- PWNs reportedly lost their pathogenicity (Cao and Han, teria from their parents or the tree host. 2001; Kawazu and Kaneko, 1997). Based on field surveys The following questions need to be addressed: 1) in China and a series of greenhouse and forest in- what are the species of bacteria on the pine-wilt affected oculations, Zhao et al. (2003) hypothesized that PWNs trees; 2) can these bacteria be carried by B. xylophilus;3) carrying phytotoxin-producing bacteria are necessary what is the role the bacteria play in PWD inductions; 4) to induce PWD. The above hypothesis has been supported what is the bacteria identity and origin. To answer some by field tests in Korea with a bactericide and a nemati- of the above questions bacteria from a P. thunbergii forest cide to control PWD (Hyeok et al., 2010). in Jiangsu Province, China were isolated, grown in pure Field tests (Tamura, 1983) showed that inoculation of culture, and mixed with axenic PWNs and the PWN- 10, 3-year-old Japanese black pine seedlings with 19,000 bacterium inoculum was inoculated into axenic, black aseptic PWN per seedling resulted in 90% tree mortality pine seedlings. Following inoculation, seven bacterial 27 to 95 days after inoculation. Kawazu (1997) assumed strains were isolated and purified from the dead seed- that the above PWN inoculum became contaminated lings. Additional seedling inoculations and identifica- with toxin-producing bacteria during inoculation which tion of the bacterial isolates were done to determine the was done under field conditions and thus, pathogenicity role of the bacteria in PWD. was attributable to these contaminants. However, Zhao and Lin (2005) reported a mutualistic symbiosis between MATERIALS AND METHODS PWN and its associated pathogenic bacteria which might explain how the mycotrophic PWN could gradually Isolation of bacteria from the bark of forest-grown, black pine: Bacteria used in our experiments were isolated Received for publication March 14, 2011. from healthy, black pine trees, P. thunbergii, growing in Department of Forest Entomology and Pathology, Nanjing Forestry Univer- a 10,000 ha suburban forest in Nanjing, Jiangsu Prov- sity, Nanjing 210037, People’s Republic of China. We thank Dr. Jack Sutherland, Victoria, BC, Canada, for editing the manu- ince, China. Twenty, 1x1cm, blocks of bark were col- script and for his constructive comments. The work was supported by the State lected from 10 random trees, 8-20 years old, DBH: Forestry Administration, People’s Republic of China (No: 2011-50). E-mail: [email protected] 6-16 cm, and washed with 100 ml of sterilized water This paper was edited by Ekaterini Riga. and filtered through a filter paper (qualitative filter 129 130 Journal of Nematology, Volume 43, Nos. 3–4, September–December 2011 paper, Grade 114v, 125 mm, Whatman Inc.) and centri- on the inoculated seedlings were recorded every day fuged (CS150GXL, HITACHI, Japan) at 40,000g for 15 using the scale: 0: no needles showing color change; min. The supernatant was discarded and sterile water 1: any needle on the seedling showing yellowing or was added to the sediment to bring the volume to 1 ml. browning for 1/4 its length; and 2: as for 1, but any PWN collection and production of axenic cultures: Bursa- needle on a seedling showing these symptoms for phelenchus xylophilus individuals were obtained from a 1/4 -1/2 its length; 3: as for 1, but any needle on naturally PWD-affected black pine from a forest in Con- a seedling showing these symptoms for 1/2 -2/3 its ghua, Guangdong Province, China, and a xylem sample length; 4: any needle of the seedling shows yellowish or was cut into 232310 mm chips. Twenty grams fresh brown more than 2/3 its length. The mean disease weight of those chips were used to extract the nematodes ranking = total disease level in the seedlings of each using a Baermann funnel (Han et al., 2003). The nema- treatment / the number of seedlings in the treatment. todes were propagated on Botrytis cinerea fungus growing Isolation and purification of the bacterial isolates from on potato-dextrose-agar (PDA) medium in Petri plates. diseased seedlings: Each dead pine seedling from the above The nematodes were washed off the PDA with sterile experiment ‘‘Inoculation experiment with axenic PWNs water and a single female nematode with eggs was ob- (ABX) + bacteria from black pine bark’’ was cut into 3 tained using a dissecting microscope. Specimens identi- mm-long segments and these were put onto NB medium fied as B. xylophilus were propagated on B. cinerea growing in Petri plates and incubated at 28 8C. The bacterial col- on PDA in individual slant cultures in 2x19 cm test tubes. onies that grew along the trails of the nematodes that Two PWN juveniles were picked up by a micro-syringe emerged from the segments on the NB medium were under a microscope and transferred to each tube and the selected according to colony shape, color and diameter tubes incubated at 28 0C for 7-10 days to let juveniles and re-cultured at 28 8Cfor2-3daysonNBmediumfor develop and reproduce; PWN reproduction occurred in subsequent purification and identification. about half of the tubes. Culturing was repeated until Toxicity of the cell-free filtrate from the cultures of bacteria sufficient nematodes were available for subsequent use. isolated from black pine seedlings: Ten ml of the purified Twenty thousand PWNs in 2 ml of 0.3% sodium carboxyl bacterial isolates, 1 x 106 cells/ml, were pipetted into methylcellulose were slowly pipetted into an acid burette 200 ml of NB liquid medium which was then shaken at containing 50 ml of 0.3% sodium carboxyl methylcellu- 110 rpm at 28 8C for 6 days. Next, sterilized water was lose. Then the burette was kept in 28 0Cfor12hrwhen added to the culture medium to adjust the bacterial the liquid containing the PWNs was drawn down and 5 ml concentration to 1x107 cells/ml which was then passed aliquots were pipetted into each of 10 test tubes. The through a bacterial filter (ME25STL, Whatman Inc). liquid in six such tubes, microscope observation showed Two ml of the medium filtrates were added to a 0.75x4.0 to contain only PWN juveniles, were combined and cm test tube and an aseptic seedling with a cut root was centrifuged at 800 g for 5 min. The 5 ml of precipitate inserted into the tube and kept under aseptic condi- with nematodes was washed five times with 5 ml sterilized tions. Liquid NB medium alone and sterilized water were 0.9% NaCl. The juveniles were sterilized with 3% H2O2 used as the two control treatments. The inoculated at 4 8C for 1 hr followed by five washings with sterilized seedlings were then incubated at 28 0Cunderaseptic 0.9% NaCl. The aseptic nematodes were transferred to conditions. Nine seedlings were used for each treatment. cultures of B. cinerea and kept for 3-4 days before use. To The symptoms were recorded every day according to the verify that the nematodes were aseptic, a few nematodes above symptom classification in ‘‘Inoculation experi- were put onto NB medium for 24 hr at 308Candsub- ment with axenic PWNs (ABX) + bacteria from black sequently checked for bacterial growth; any non-aseptic pine bark’’.