Pathogenicity of Aseptic Bursaphelenchus xylophilus

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.


Introduction
Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Although the first occurrence of pine wilt disease (PWD) was reported in 1905 in Japan [1], PWN was not identified as the causal agent of the disease until 1971 [2]. PWD has spread to other Asian countries such as China and Korea and to Europe (Portugal) [3,4,5,6], and has become a worldwide threat to pine forests and forest ecosystems with great economic losses. PWD kills 1,000,000 m 3 of pine trees annually in Japan [7], and had damaged approximately 7,811 ha of pines in Korea by 2005 [8]. The direct economic losses caused by PWD in China were estimated at approximately $300 million with indirect economic losses exceeding $3 billion [9].
Despite the significance of this disease, the pathogenic mechanism of PWD remains to be elucidated. Until recently, PWN was believed to be the only pathogenic agent causing the disease [10,11,12,13,14]. Because the physiological and histological changes of diseased trees occur before a rapid increase in the population of nematodes, Oku et al. proposed that other agents might be involved in the pathological process [15]. The possible association of PWN and toxin-producing bacteria was reported later [16]. More recently, bacteria from various genera have been found to be associated with B. xylophilus [17,18,19,20,21,22]. Certain inoculations indicated that bacteria carried by the PWN may play an important role in the pathogenicity of the disease. For example, Kawazu and Kaneko reported that the asepsis of the PWN caused it to lose its pathogenicity [23]. Han et al. reported that inoculating aseptic black pine seedlings with aseptic PWNs or bacteria alone did not lead to browning or wilting, but inoculation with aseptic PWNs combined with the bacteria isolated from PWN resulted in the onset of severe symptoms [19]. Zhao et al. discovered that inoculation with bacteria alone did not lead to the development of disease symptoms, but a combination of axenic PWNs and bacteria led to disease, while seedlings exhibited none or weak symptoms when inoculated with axenic PWNs or axenic PWNs combined with the non-pathogenic bacterium [20]. The results led the authors to propose a new hypothesis that PWD was a complex disease induced by both, PWNs and their associated pathogenic bacteria.
Although the inoculation tests outlined above provide apparently convincing evidence of the role of bacteria, some scientists think that a note of caution needs to be applied, as bacteria alone cannot cause the disease [24]. It has been proposed that bacteria associated with PWNs are chance contaminants. Because bacteria exist both inside and outside of the host tree, they are not pathogenic [14]. Therefore, further research in this area is needed. The objective of the present study was to evaluate the pathogenicity of bacteria-free PWNs on axenic plantlets derived from clonal plants of Pinus densiflora and seven-month-old P. massoniana aseptic seedlings.

Pine Wood Nematode (Non-aseptic)
Four isolates of B. xylophilus (strongly virulent AMA3c1, AN19 and AA3, and weakly virulent YW4) were used in all experiments. The strain AMA3c1 was an inbred line maintained by Lihua Zhu in Jianren Ye's laboratory for 20 generations of full-sibling mating [25]. It was derived from the wild isolate AMA3. All of the wild isolates were established with 20-30 individuals collected from dead pine trees by Huang Ren-e [26]. The origin of the nematode isolates is listed in Table 1. The nematodes were subcultured on the fungus Botrytis cinerea in a 25uC incubator for further use.

Acquisition of Bacterium-free Nematodes
Populations of the four isolates were multiplied on B. cinerea cultures on potato-dextrose-agar (PDA) medium at 25uC for 7 days, then washed onto a piece of filter paper in a Baermann funnel with sterile water and kept there for 12 h. A volume of 10 mL of nematode-containing liquid was collected from the bottom of the funnel and centrifuged at 2919 g for 5 min. The supernatant was discarded and the nematode-containing precipitate was washed several times and resuspended with sterile water. The nematode suspension was poured onto a sterilized cover slip in a petri dish, incubated at 25uC and allowed to lay eggs for 4-6 h, after which the nematodes were discarded. Eggs adhering to the surface of the cover slip were rinsed several times with sterile water to remove the nematodes, and the cover slip was placed in a petri dish containing 15% H 2 O 2 for 60 min at 25uC. The cover slip was rinsed 3 times with sterile water and placed at 25uC in the dark on the mycelia of B. cinerea grown on the PDA medium. To prevent the eggs from drying out, a piece of sterile wet absorbent cotton was placed on the cover slip. This method was used for a total of 12 populations, 3 from each of the 4 isolates used for the experiment. The propagated nematodes were collected with a Baermann funnel under aseptic conditions and were checked for the presence of bacteria by culturing them in nutrient broth (NB) (3 g/L beef extract, 5 g/L peptone and 5 g/L NaCl, pH 7.2 ) liquid medium in a flask for more than 7 days.

Isolation and Identification of Bacteria from Pine Wood Nematodes
Bacteria were directly isolated from AMA3c1 that had been incubated on a lawn of B. cinerea PDA medium at 25uC for 7 days. After harvesting the pine wood nematodes with 10 mL of sterile distilled water per plate, the suspension was centrifuged at 2919 g for 5 min. The supernatant was discarded and the pellet was washed 3 times with sterile distilled water. One mL of the suspension (ca. 20000 nematodes) was ground with a mortar and pestle, and a 1 mL aliquot was removed and serially diluted with sterile distilled water. One hundred mL of each dilution was spread onto NB agar medium and incubated at 30uC for 3 days. Colonies were selected and then further cultured to establish pure cultures. Two bacterial strains were isolated from AMA3c1, and were designated as AMA3c1-1 and AMA3c1-2.
The isolated bacteria were mainly identified on the basis of 16 S rRNA gene sequencing. Total genomic DNA was extracted as described by Sambrook et al. [27]. From the extracted genomic DNA, 16 S rRNA genes were PCR amplified with the bacterial universal primers 27F and 1492R. PCR was performed in the following program: one cycle of 5 min at 94uC, followed by 30 cycles of 30 s at 94uC, 30 s at 56uC, and 1 min at 72uC, and one cycle of 5 min at 72uC [28]. PCR products were purified using the TaKaRa DNA Purification Kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions and cloned into the pMD19-T vector (TaKaRa), transformed into Escherichia coli JM109, extracted, amplified and purified according to standard procedures, and then sequenced at Spring Ltd. (Nanjing, China). The 16 S rRNA gene sequence similarity was determined using the EzTaxon server [29]. The 16 S rRNA gene sequences were deposited in the NCBI database. The phylogenetic dendrograms were constructed by the neighbor-joining method using the molecular evolutionary genetics analysis (MEGA) software version 4.0.

Culture of Aseptic P. massoniana Seedlings
Mature seeds of P. massoniana were soaked in 0.1% KMnO 4 solution for 12 h and then washed in tap water for 1 h; floating seeds were then discarded. The seed coats of the remaining seeds were removed with sterilized forceps and intact megagametophytes were surface sterilized with 70% ethanol for 30 s, and then soaked in 0.1% aqueous HgCl 2 (w/v) for 3 min followed by 3 rinses with sterile water. Megagametophytes were then placed on a PDA plate for accelerated germination at a certain distance from each other. The process of accelerated germination was monitored frequently and uncontaminated germinated megagametophytes were transferred to a 500 mL flask containing 200-250 mL Gresshoff and Doy [30] medium (GD) supplemented with 0.5-1.0% activated charcoal and cultured there for 7 months under controlled conditions (temperature 2562uC, 16 h of light at 2000 lx).

Culture of Aseptic Microcuttings of P. densiflora
The culture of aseptic microcuttings of P. densiflora was performed as described by Zhu et al. [31]. Cotyledon-hypocotyl explants obtained from 21-28 days old aseptically grown seedlings of P. densiflora were cultured on GD medium containing 4.0 mg/L 6-BA (6-benzylaminopurine) and 0.1 mg/L NAA (a-naphthylacetic acid) for 5 weeks for bud induction. Induced axillary buds were subcultured on GD medium supplemented with 0.5-1.0% activated charcoal for elongation. Elongated shoots (10-20 mm long) were excised and cut into 5-8 mm stem sections and then cultured on GD medium supplemented with 2.0 mg/L 6-BA and 0.2 mg/L NAA for proliferation. After 4 weeks of culture, shoots were transferred to GD medium supplemented with 0.5-1.0% activated charcoal for elongation. The shoots were subcultured into fresh medium at one month intervals. All cultures were maintained under the same conditions used for the culture of P. massoniana seedlings. All shoots generated from a single seed were identified with the same clonal number.

Inoculation
Preparation of inoculum. The aseptically cultured nematodes were harvested by Baermann funnel under aseptic conditions and the suspension was adjusted to a concentration of 10000 nematodes/mL. One mL of this suspension was cultured in NB liquid medium in a flask for more than 7 days at 30uC to assess for contamination, and the remainder was maintained at 4uC. After verifying that the nematodes were bacteria-free, the suspension at 4uC was used for the inoculation test. The unsterilized nematodes cultured on B. cinerea for 5-7 days were harvested 1 day before inoculation and adjusted to the same concentration as that of the aseptic nematodes. The bacterial isolates obtained from the nematodes were used to test their pathogenicity. The two bacterial suspensions that had been shake-cultured in a 200-mL flask for 2 days at 28uC were adjusted to a concentration of 2610 6 bacteria/mL and then mixed in equal volumes.
Inoculation of aseptic microcuttings with AMA3c1 and bacteria. Aseptic microcuttings elongated on GD medium for 3 months were collected under aseptic conditions and the shoot tips were cut away. The treated microcuttings were placed into fresh medium, a piece of aseptic absorbent cotton was placed on the wound, and 0.02 mL of the inoculum suspension liquid containing 200 nematodes or 4610 4 bacteria (the mixture of two bacterial suspensions) was added to the cotton. Inoculation with sterile water was used as a control. Each treatment was performed 3 times using a minimum of 10 microcuttings. All cultures were then maintained at 30uC under continuous, cool white fluorescent illumination (2000 lx) with a 16 h photoperiod. The number of wilted seedlings was recorded every 2 days until 20 days after inoculation.
Inoculation of aseptic seedlings with AMA3c1. The shoot tips of 7-month-old P. massoniana aseptic seedlings (10-15 cm tall) were cut away with sterilized scissors under aseptic conditions, and a piece of aseptic absorbent cotton was placed on the wound. Twenty five mL of the inoculum suspension liquid containing 250 nematodes were added to the cotton. Inoculation with sterile water was used as a control. Each treatment was performed using at least 10 seedlings. The treated cultures were then maintained under the same conditions described above. The number of wilted seedlings was recorded every 2-days until 30 days after inoculation.
Inoculation of aseptic microcuttings with AMA3c1, AN19, AA3 and YW4. Aseptic microcuttings elongated on GD medium for 6 months were collected under aseptic conditions, and the shoot tips were cut away. The treated microcuttings were placed into fresh medium, a piece of aseptic absorbent cotton was placed on the wound, and 0.025 mL of the inoculum suspension liquid containing 250 nematodes was added to the cotton. Inoculation with sterile water was used as a control. Each treatment was performed using 10 microcuttings. All cultures were maintained under the same conditions as above. The number of wilted seedlings was recorded every 2-days until 20 days after inoculation.

Recovery of the Nematode from the Wilted Microcuttings
Twenty or 30 days after inoculation, wilted microcuttings or seedlings were removed from the bottle and cut into 2-4 mm sections with aseptic scissors; nematodes were extracted with a Baermann funnel. After 12 h, nearly 1 mL of nematode-containing liquid at the bottom of the funnel was collected. The nematodes from the microcuttings inoculated with aseptic nematodes was recovered under aseptic conditions. The recovered nematodes were assessed for bacterial contamination by culturing 200 mL of each suspension in NB liquid medium at 30uC for more than 7 days. The number of recovered nematodes was counted under the microscope.
Statistical Analysis SPSS 13.0 software (SPSS, Inc., Chicago) was used for variance analysis. All the data were expressed as the mean 6 standard deviation.

Acquisition of Bacterium-free Nematodes
Nematode eggs were placed on the mycelia of B. cinerea after surface sterilization with H 2 O 2 . After approximately 2-3 weeks, the fungal mats disappeared from the plate. The nematodes were extracted and the results of surface sterilization were assessed. Bathing PWN eggs in 15% H 2 O 2 for 60 min was shown to lead to complete asepsis. A total of 12 populations were assessed in NB liquid medium for more than 7 days, and only one population from AA3 was not bacterium free.

Identification of Bacteria
The 16S rRNA gene sequence analysis identified strains AMA3c1-1 and AMA3c1-2 as Pseudomonas sp. and Rhizobium sp., respectively. The 16S rRNA gene sequences of these two bacterial strains were deposited in the NCBI database, and the accession numbers were JQ419489 and JQ419490, respectively.

Pathogenicity of Aseptic AMA3c1 on Aseptic P. densiflora Microcuttings
The shoot tips of aseptic microcuttings from clones 10-4, 16-2 and 1-A of P. densiflora were cut down and inoculated with aseptic AMA3c1, unsterilized AMA3c1 and the mixture of the two strains of bacteria. Microcuttings inoculated with aseptic AMA3c1 and with unsterilized AMA3c1 wilted, whereas those inoculated with bacteria and the control survived for 20 days after inoculation (Figure 1 A & Figure 1 B, Table 2). There were no differences in wilting symptoms between the microcuttings treated with aseptic nematodes and those exposed to unsterilized nematodes. In the case of clone 10-4, 4 of 18 microcuttings wilted 6 days after inoculation with aseptic AMA3c1 and another 13 microcuttings wilted during the following 20-day period. In the case of clone 16-2, 6 of 20 microcuttings wilted 6 days after inoculation with aseptic AMA3c1 and another 14 microcuttings wilted 14 days after inoculation. In clone 1-A, the presence of bacteria caused earlier symptoms, but aseptic nematodes eventually wilted 80% of the microcuttings over a 42-day period.

Pathogenicity of Aseptic AMA3c1 on Aseptic P. massoniana Seedlings
In 7-month-old aseptic P. massoniana seedlings, inoculation with PWNs caused typical pine wilt symptoms, while plants inoculated with sterile water remained healthy ( Figure 2). Eighteen of the 20 seedlings inoculated with aseptic AMA3c1 wilted within 30 days after inoculation, and the fastest wilt occurred 6 days after inoculation. Eight of 10 seedlings wilted after inoculation with unsterilized AMA3c1 (Table 3). These results suggest that asepsis of the inbred strain AMA3c1 did not result in the loss of pathogenicity.
The nematodes recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs were cultured in NB liquid medium at 30uC for more than 7 days and no microorganisms grew on the medium.

Discussion
PWD is a very complex disease, and the role of bacteria in the pathogenesis of PWD is controversial. Although inoculation experiments performed under sterilized conditions [19,20,23] and in the field [20,32] showed that surface sterilized PWNs lose their pathogenicity, Tamura reported that 3-year-old P. densiflora inoculated with aseptic nematodes wilted [33]. Bolla and Jordan also reported aseptic PWNs showed no alterations in their pathogenicity against 45-day-old P. sylvestris [34]. Kawazu and Kaneko speculated that the different results of field inoculations might be due to the fact that axenic PWNs regain their pathogenicity after being re-contaminated with bacteria during inoculation [23]. Because bacteria exist everywhere in uncontrolled conditions, in vitro inoculation using aseptic nematodes, purified bacteria and hosts grown aseptically was more suitable for the accurate study of plant-pathogen interactions.
Obtaining aseptic nematodes was the first crucial step for the accurate study of the interaction between parasitic nematodes and plants. The successful disinfection of PWNs has been achieved using various chemical disinfecting solutions and different types of sterilizing protocols [19,34,35,36]. Because PWNs carry different bacteria on the surface of their body, it is difficult to obtain axenic PWNs. The present study used a simple and efficient method to obtain bacteria-free PWNs that consist of bathing the PWN eggs during the 4-6 h incubation and allowing them to adhere on the cover slip in 15% H 2 O 2 for 60 min.  Table 3. Wilting ratios of 7-month-old P. massoniana aseptic seedlings inoculated with aseptic AMA3c1 and unsterilized AMA3c1, and number of nematodes recovered from the seedlings.

Inoculum
Wilting ratio (%) Two bacterial strains were isolated and identified as Pseudomonas sp. and Rhizobium sp. from AMA3c1. According to the literature, the PWN carries bacteria from different genera and the bacterial communities differ among countries. Bacillus is predominant in Japan [37], Pseudomonas, Pantoea, Stenotrophomonas in China [19,20,38], Burkholderia, Brevibacterium, Ewingella, Enterobacter, Serratia in Korea [21], and Burkholderia, Pseudomonas and other bacteria from Enterobacteriaceae are mainly found in Portugal [22,39]. Although part of the Chinese PWN population was introduced from Japan [40], Bacillus was not reported in China except in the work of Tan and Feng [41]. Bacillus was also not found in Portugal [22]. Although Pseudomonas has been reported as one of the major phylogenetic groups in Portugal and China, it does not indicate that a Pseudomonas species is associated with PWN in Portugal  Table 4. Wilting ratios of tissue-cultured microcuttings of Pinus densiflora inoculated with AMA3c1, AN19, AA3 and YW4, and number of nematodes recovered from the microcuttings. because none of the isolated Pseudomonas species was found in all sampling areas and they have not been detected in surfacedisinfected nematodes by using molecular techniques [22]. The difference of bacterial composition was likely owing to the discrepancy of geographic areas, environmental factors, pine hosts, and vectors [42]. For example, P. massoniana is the dominant host in China, P. thunbergii is the dominant host in Japan, and P. pinaster is the dominant host in Portugal.
In vitro experiments showed that inoculation of pine microcuttings and seedlings with bacteria alone did not cause disease, which was in accordance with previous reports [17,19,20]. However, aseptic PWNs successfully infected in vitro-grown pine microcuttings and seedlings and caused typical pine wilt symptoms. In our first experiment, inoculation of 3-month-old microcuttings derived from tissue-cultured P. densiflora with the aseptic inbred nematode AMA3c1 caused wilting. The results were further confirmed by the inoculation of 7-month-old P. massoniana aseptic seedlings with aseptic AMA3c1, and microcuttings of P. densiflora with aseptic AN19, AA3 and YW4. These results were contrary to those of Kawazu and Kaneko [17], Han et al. and Zhao et al. [19,20], who reported that seedlings inoculated with axenic nematodes did not show wilting symptoms or a few showed only minor symptoms of stem shrinkage without needle browning. One possible reason for these discrepancies may be that the observation period in the studies of Han et al. and Zhao et al. was too short (only 4 days) for the appearance of wilt symptoms [19,20]. Tamura reported that most of the aseptic P. thunbergii seedlings inoculated with aseptic nematodes collapsed, and the fastest wilt occurred 5 days after inoculation with 1500 nematodes on callus tissues formed on seedling root tips [30]. In the present study, the fastest discoloration of leaves or shrinkage of stems was usually observed 6 days after inoculation.
The nematodes were recovered from wilted microcuttings and seedlings that had been inoculated with either aseptic nematodes or unsterilized nematodes, and the asepsis of nematodes recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs was reconfirmed. Surprisingly, the number of recovered nematodes from wilting microcuttings and seedlings inoculated with aseptic PWNs was significantly higher than that from plants inoculated with unsterilized PWNs. Similar observations have previously been reported by Tamura [30]. When the root callus was inoculated with the combination of aseptic nematodes and bacteria, the number of nematodes decreased rapidly and they did not propagate over a 55-day period. It is likely that bacteria killed callus tissues before the reproduction of nematodes [30]. Despite the successful recovery of nematodes from wilting seedlings in the in vitro inoculations reported by Kawazu et al., Han et al. and Zhao et al. [43,19,20], these authors did not report on the number of nematodes recovered, and it is therefore not possible to determine whether any differences were present. However, Zhao et al. [20] performed field inoculation and reported that the number of nematodes per gram of wood in pine trees inoculated with axenic PWNs was lower than that of trees inoculated with axenic PWNs combined with the pathogenic bacterium or with wild PWNs in the control. The reason for the inhibition of nematode propagation in the present study is not clear, and further investigation is therefore required.
In conclusion, the present study demonstrated that inoculation with axenic PWNs caused wilting of young pine microcuttings and seedlings. However, since there are significant differences between the physiology and pathology of adult pine trees and young seedlings, further research on the role played by nematodes and associated bacteria in disease development should be conducted using axenic PWNs and at least two-or three-year-old pine seedlings grown aseptically.