Correlation between acoustic emission, water status

Tree Physiology 27, 969–976 © 2007 Heron Publishing—Victoria, Canada

Correlation between acoustic emission, water status and xylem embolism in pine wilt disease KENJI FUKUDA,1,2 SHIN UTSUZAWA3–5 and DAISUKE SAKAUE6 1

Laboratory of Evaluation of Natural Environment, Institute of Environmental Studies, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8563, Japan

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Corresponding author ([email protected])

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MRTechnology, Inc., 169-1 Kouya Tsukuba-city Ibaraki, 300-2642, Japan

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Institute of Applied Physics, University of Tsukuba, Ibaraki, Japan

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Present address: New Mexico Resonance, 2301 Yale Blvd. SE, Suite C-1, Albuquerque, NM 87106, USA

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University Forest at Tanashi, University of Tokyo, Tokyo, Japan

Received January 6, 2006; accepted November 9, 2006; published online April 2, 2007

Keywords: Bursaphelenchus xylophilus, cavitation, MRI, nondestructive observation, pinewood nematode, Pinus thunbergii, ultrasonic acoustic emission.

Introduction Despite heated debate (e.g., Zimmermann et al. 1994, Canny 1995), the cohesion–tension (C–T) theory (Böhm 1893, Dixon 1914) is the most widely accepted model for water transport in terrestrial plants (Tyree 1997). According to C–T theory, xylem water in plants is under significant negative pressure and is therefore vulnerable to cavitation (Pockman et

al. 1995). The C–T theory predicts that, when tissue water potentials are low, trees operate near the point of catastrophic xylem dysfunction by cavitation in vessels and tracheids. Cavitation in a vessel or tracheid occurs as a result of “air seeding” from neighboring conduits through pit pores when the stem water potential decreases below a critical threshold (Sperry and Tyree 1988). If the water potential in the stem decreases below this threshold, cavitation in the stem xylem will increase resistance to water flow. If transpiration from the leaves is maintained, the increase in resistance will result in a further decrease in water potential and induce more cavitations. This so-called runaway embolism (Tyree and Sperry 1988) is usually prevented by stomatal closure, which minimizes transpiration and keeps water potential above the critical threshold (Nardini and Salleo 2000, Salleo et al. 2000). Pine wilt disease, caused by the pinewood nematode (Bursaphelenchus xylophilus (Steiner et Buhler) Nickle), is one of the most devastating tree diseases, and the symptoms characterizing its development have been intensively studied (Fukuda 1997). In the early stage of the disease, tracheids and neighboring xylem resin canals become embolized. The underlying mechanisms have yet to be definitively established, but suspected causes of cavitation include leakage of oleoresin from the resin canals to the tracheids (Sasaki et al. 1984), and exudation of terpenoids (Kuroda 1989, 1991) and vacuole contents from xylem ray parenchyma cells (Nobuchi et al. 1984), which decrease pit conductivity and water potential. Increases in pit pore size caused by cellulase activity (Odani et al. 1985) or benzoic acid synthesized in infected host tissue (Ikeda et al. 1989) and excretion of surface-active substances by the nematodes (Sakaue et al. 1999) are also hypothesized to induce cavitation. In the advanced stages of the disease, abrupt decreases in water potential resulting in death are induced by cavitation in the stem xylem (Fukuda et al. 1992, Ikeda and Kiyohara 1995, Ikeda 1996). Cavitation events in xylem conduits have been studied by

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Summary The occurrence of cavitation events and embolism during the latent, early stage and the late developmental stages of pine wilt disease was monitored nondestructively by acoustic emission (AE) and high-resolution magnetic resonance microscopy, respectively, and the results were compared with changes in leaf water potential and stem thickness. In the latent stage of the disease, when no embolisms were observed, cavitation events were detected by AE during the daytime in water-stressed Japanese black pine (Pinus thunbergii Parl.) seedlings, indicating that cavitation occurred at the individual tracheid level. In the early stage of the disease, an increase in the frequency of AE events occurred coincidentally with the occurrence of patchy embolisms at the mass tracheid level. The threshold water potential for such mass cavitation was higher than that causing cavitation of individual tracheids during the latent stage of the disease. In the advanced stage of the disease, explosive AE events were observed coincidentally with drastic enlargement of embolized areas and decreases in water potential. The AE events in the latent stage occurred only during the daytime, whereas, in the early and advanced stages of the disease, they also occurred at night. The explosive occurrence of cavitation in the advanced stage was thought to be a case of “runaway embolism.”

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FUKUDA, UTSUZAWA AND SAKAUE

RF shield box made of 0.5-mm-thick brass plates that could be opened or closed. The MR microscope consisted of a four-column permanent magnet with a field strength of one Tesla and a 6-cm air gap (Neomax Co. Ltd., Japan) and a portable magnetic resonance imaging console (MRTechnology Inc., Tsukuba, Osaka, Japan). A pair of flat gradient coils was fixed onto the poles of the magnet. Sunlight or auxiliary illumination during the daytime was supplied from the open top and both sides of the magnet, and the pot was watered when the soil surface was dry. We used T1-weighted spin-echo sequences with a repetition time (TR) of 500 ms and an echo time (TE) of 22 ms to obtain 2-D images (256 × 256 pixels) of the water content in a transverse section of the pine stem. The values of TR and TE were determined in preliminary experiments to produce images of cavitation in xylem with the highest contrast. Proton density sequences with longer TRs are usual when imaging water in living organisms, however, not only water in conducting xylem but also resinous materials in embolized xylem produce strong signals in the proton density sequence because resins contain protons. With a shorter TR, signals from the resinous materials become negligible (Utsuzawa 2006). Therefore, a T1-weighted image with a short TR was most suitable for differentiating between functional and embolized xylem in our system. Xylem embolisms visualized in these seedlings by MR microscopy were reported by Utsuzawa et al. (2005). To re-estimate the sizes of the embolized areas more precisely than was done by Utsuzawa et al. (2005), we compared each MR image with an MR image of the same section of the xylem taken when healthy on either March 3 or August 13, when no embolisms were evident. By overlaying the two images with image analyzing software (Adobe Photoshop for Windows Ver. 5, Adobe Systems Inc.), we were able to detect embolized xylem areas based on a brightness difference threshold for pixels that changed from bright to dark. The areas of the embolisms were then calculated with the free software LIA32 Ver. 0.376β1 (www.agr.nagoya-u.ac.jp/%7Eshinkan/LIA32/index-e.html). An ultrasonic sensor (AE901U, NF-electronics Co., Tokyo, Japan), which was fixed on the head of an iron pushpin with

Materials and methods Two potted 3-year-old Japanese black pine seedlings were inoculated with 10,000 pinewood nematodes of a virulent isolate (S10) by dropping 0.01 ml of an aqueous nematode suspension into a wound on a current-year shoot. The wound was made with a razor blade and was deep enough to reach the xylem surface. Seedling 1 was inoculated on March 1, 2004, and Seedling 2 was inoculated on August 12, 2004. The same seedlings were referred to as Seedling 2 and Seedling 3, respectively, by Utsuzawa et al. (2005). Seedling height was about 60 cm, and basal diameter was 10 mm in Seedling 1, and 12 mm in Seedling 2. After inoculation, the water content in the xylem tracheids of a 1-year-old stem was monitored daily with a compact MR microscope (Figure 1). An eight-turn radio-frequency (RF) coil (10-mm inner diameter) was wrapped around the pine stem 15 cm above the soil surface. The coil was installed in an

Figure 1. Compact magnetic resonance microscope system used to measure xylem tracheid water content. Seedling height was 60 cm. Abbreviations: RF, radio frequency; and BNC, Bayonet Neill Concelman (co-axial).

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detecting sonic or ultrasonic acoustic emissions (AEs) from the xylem of stems, branches and leaves (Milburn 1973, Tyree and Dixon 1983, 1986, Tyree and Sperry 1989, Ikeda and Ohtsu 1992, Kikuta et al. 1997). Acoustic emissions occur concurrently with decreases in plant water potential, losses of xylem density, decreases in hydraulic conductivity and decreases in the area of conducting xylem that stains with dye (Dixon et al. 1984, Tyree et al. 1984, Jones and Peña 1986, Lo Gullo and Salleo 1991). The abrupt decrease in water potential in trees affected by pine wilt disease accompanies intensive ultrasonic AE events (Ikeda 1996, Kuroda 1996), which are thought to indicate runaway embolism (Ikeda 1996), a phenomenon not normally observed in healthy plants (Tyree and Sperry 1988). In pine wilt disease, the cavitation process cannot be followed with destructive methods, such as staining the xylem with water-soluble dyes or measuring the conductivity of stem segments (e.g., Kuroda et al. 1988, Fukuda et al. 1992, Ikeda 1996). Recently, new methods for studying water transport in xylem have been proposed, including some, such as magnetic resonance imaging, that are nondestructive. High-resolution magnetic resaonance imaging (MR microscopy) allows observation of sap flow in plant tissue (Johnson et al. 1987, Bentrup 1996, Koeckenberger et al. 1997, Rokitta et al. 1999, Ishida et al. 2000, van der Weerd et al. 2001, Scheenen et al. 2002) and has been used to visualize cavitation in xylem vessels in vivo (Holbrook et al. 2001, Clearwater and Clark 2003, Kuroda et al. 2006). Previously, we monitored cavitation in pine wilt disease with a compact MR microscope system and demonstrated that rapid enlargement of the embolized area occurs in the advanced stage of pine wilt disease (Utsuzawa et al. 2005). To clarify the mechanism of cavitation and embolism in pine wilt disease, we measured the water status of Japanese black pine (Pinus thunbergii Parl.) seedlings by changes in leaf water potential and relative stem diameter and compared these changes with the cavitation events and embolisms monitored by AE and MR microscopy, respectively.

ACOUSTIC EMISSION AND EMBOLISM IN PINE WILT DISEASE

Results Cavitation caused by injury and noises caused by insertion of the pushpins into the xylem during placement of the AE sen-

Figure 2. Placement of an acoustic emission (AE) sensor on a pine stem. Cavitation events were detected by a pushpin inserted into the xylem. The AE sensors were placed on the main stem of the pine seedlings at the stem base (Seedling 1 and 2) and on a current-year shoot (Seedling 2). Stem thickness ranged between 6 and 10 mm.

sors in Seedling 2 are shown in Figure 3. Abnormal AE events occurred during the first 2 h after pushpin insertion. Thereafter, AE events were negligible (0–5 events per 15 min). The relationship between leaf water potential and stem diameter, which were monitored simultaneously in Seedling 2 from August 17 to September 3, is shown in Figure 4. Two measurements immediately after strain gauge installation and three measurements after seedling death were omitted. Variation in relative stem diameter showed a good correlation with leaf water potential during this period, indicating that it could serve as an index of stem water potential. The MR images used to assess embolization of the stems of Seedlings 1 and 2 are shown in Figures 5 and 6, respectively. The MR images taken at half-day intervals were added to the data from Utsuzawa et al. (2005) to gain a better understanding of the course of embolism development. In the images, conducting xylem appeared as the brightest area, whereas bark, pith and latewood were less bright, and embolized xylem appeared as dark patches (Utsuzawa et al. 2005). A small patch of embolism in the xylem was first observed on March 9 in Seedling 1 and on August 19 in Seedling 2. The

Figure 3. Acoustic events after insertion of the acoustic emission (AE) sensors. Most noise and cavitation caused by injury resulting from sensor installation were observed within 4 h after sensor insertion (arrow).

Figure 4. Water potential of current-year needles and relative stem diameter (strain gauge (SG) measuring circuit output) of a 1-year-old stem at 6 cm above the soil surface in Seedling 2.

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silicone sealant, was inserted into the xylem of the main stem to monitor AEs from the xylem (Figure 2). The sensor had a resonance frequency of 140 kHz and a sensitivity of –10 dB. Each AE sensor was connected to an amplifier– discriminator (AE-Tester 9501, NF-electronics). The sensitivity of the amplifier was 50 µV, and the 1-ms open collector output was connected to a 1-kHz pulse-count data logger (LOGBOOK B5-pulse, LOG-Denshi Co., Sapporo, Japan). Data were collected every 15 min. For Seedling 1, AEs from a 3-year-old stem were monitored at the stem base, and in Seedling 2, AEs were monitored with two sensors, one placed on a current-year stem (15 cm above the portion observed by MR microscopy) and one at the stem base (15 cm below). To monitor the water status of Seedling 2, changes in diurnal relative stem diameter and needle water potential were measured. Changes in stem diameter were monitored by the strain gauge method (Ueda et al. 1996, Ueda and Shibata 2001). A 9-mm-long strain gauge of 200 kΩ (Kyowa Dengyo Co., Tokyo, Japan), connected to a strain data logger (Tokyo-GAS Frontier Institute, Tokyo, Japan), was affixed to the bark surface of the 3-year-old stem at 6 cm above the soil surface (9 cm below the MR microscope). The strain, as reflected in a change in the resistance of the strain gauge, was recorded as the output of a measuring circuit. The daily maximum needle water potential was measured with a pressure chamber (PMS-600, PMS Instruments, Corvallis, OR) around 0700 h for two or three needles of the current-year shoot of the main stem. Measurements were made at 2–3-day intervals, and the pine seedling was kept in the dark from the previous night by covering the whole seedling with a cardboard box until the measurement was performed. The daily minimum water potential fluctuates rapidly in response to climatic conditions, whereas daily maximum water potential is a good indicator of the development of pine wilt disease symptoms.

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Figure 5. Magnetic resonance images of the transverse section of a 1-year-old stem of Seedling 1 after inoculation with pinewood nematodes (repetition time = 500 ms, echo time = 22 ms). Redrawn from Utsuzawa et al. 2005, with additional images.

13 to 17, AE rates increased dramatically and simultaneously with the rapid enlargement of embolized areas in the 1-yearold stem. The relative area of embolized xylem showed a strong sigmoidal relationship with cumulative AE events (Figure 8). From the regression, AE rate plateaued at a relative embolized xylem area of 60%, indicating that the entire xylem area in the basal part of the stem of Seedling 1was not embolized (data not shown). Thus, cavitation in the upper portion released water tension in the basal portion of the stem and prevented new cavitation at the AE measurement point. When Seedling 1 was inoculated on March 1 it was not subject to water stress.

Figure 6. Magnetic resonance images of the transverse section of a 1-year-old stem of a Japanese black pine seedling (Seedling 2) after inoculation with pinewood nematodes (repetition time = 500 ms, echo time = 22 ms). The white circle indicates the first cavitation. Redrawn from Utsuzawa et al. 2005, with additional images.



period before the earliest visible embolism was regarded as the latent stage of the disease. The embolized areas increased in number and gradually enlarged and began to fuse together over several days (early stage of disease development). The embolized areas then enlarged drastically to cover the entire xylem between March 12 and 16 in Seedling 1 and between August 30 and September 5 in Seedling 2 (advanced stage of disease development). The development of embolized areas in Seedling 1 was compared with AE rates and cumulative AE events (Figure 7). Rates of AE showed small predawn peaks on March 1 and 11, when the embolized area noticeably increased. From March


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Figure 8. Relationship between embolism in a 1-year-old stem observed by magnetic resonance microscopy and cumulative acoustic emission (AE) events monitored at the base of a 3-year-old stem in Seedling 1.

For Seedling 2, leaf water potential, changes in stem thickness, AE rates, cumulative AE events and the enlargement of embolized areas are shown in Figure 9. No embolization was detected by MR microscopy until August 19. Relative stem diameter decreased during the daytime and recovered at night during the latent stage. This diurnal stem shrinkage and expansion paralleled the diurnal trend in the xylem water potential of a healthy plant (Ueda et al. 1996, Ueda and Shibata 2001). During the latent stage of the disease, leaf water potential remained above –1.0 MPa, and AE events were detected only

during the daytime. From August 20 to 29, AEs in both the current shoot and the stem base showed small peaks during the daytime, and patches of xylem embolism were observed by MR microscopy. During this early stage of the disease, AE peaks were higher on days when stem shrinkage was greatest. Leaf water potential began to decrease after August 30 and reached –1.5 MPa on September 2. This coincided with drastic stem shrinkage during the daytime on September 1, which was not followed by overnight recovery, indicating the start of the

Figure 9. (a) Maximum water potential of leaves and relative stem diameter (strain gauge (SG) measuring circuit output), (b) acoustic emission (AE) rates and (c) cumulative AE events and embolism estimated from magnetic resonance microscopy in Seedling 2.



Figure 7. Enlargement of embolized areas in pine xylem and acoustic emission (AE) events in Seedling 1 after inoculation with pinewood nematodes.

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Discussion This is the first report of concurrent monitoring of cavitation and embolism by AE and MR microscopy, respectively. We found low rates of AEs during the daytime in the latent stage of infection with pine wilt disease, which were unaccompanied by xylem embolism at a scale observable by MR microscopy. This indicates that daytime cavitation due to water stress in healthy pine seedlings does not occur in a mass of tracheids, but only in individual tracheids, because the resolution of the

Figure 10. Embolism in a 1-year-old stem observed by magnetic resonance microscopy and cumulative acoustic emission (AE) events monitored in a current-year shoot and at the base of a 3-year-old stem in Seedling 2.

MR microscope was 40 µm, whereas the radii of pine tracheids range from 8 to 60 µm. The AEs occurred more frequently when patches of embolism were observed in the early stage of the disease. This phenomenon accords with earlier findings (Ikeda 1996, Kuroda 1996). In the early stage of the disease, the frequency of AEs was correlated with decreased water potential during the daytime in Seedling 2, and this phenomenon differed from cavitation in individual tracheids of healthy plants because it was accompanied by patchy embolism. Moreover, this phenomenon occurred at night in Seedling 1. The occurrence of AE events at nighttime was also reported by Kuroda (1996). The occurrence of AE events at nighttime in Seedling 1, but not in Seedling 2, during the early stage of the disease might be the result of a difference in the water status of the seedlings. Seedling 1 was inoculated in March when current-year shoots had not yet expanded and transpiration would have been much lower than at the same stage of infection in Seedling 2. The threshold change in relative stem diameter (estimated by change in strain gauge resistance) for cavitation during the early manifestation of infection seemed to differ from that during the latent stage (Figure 11). In the latent stage, the threshold strain gauge measuring circuit output was around 100 mV, indicative of a tissue water potential of –1.1 MPa, whereas in the early and advanced stages, it was around 120 mV, indicative of a tissue water potential of –0.6 to –0.7 MPa. This suggests that patchy embolism in the early manifestation of the disease is facilitated by decreased water potential in summer, whereas enlarged areas of xylem embolism in the late stage of the disease are the result of a pathological change. Differences in the threshold water potential for cavitation during the development of pine wilt disease have also been reported by Ikeda (1996) and are thought to be caused by the secretion of substances from resin canals or ray parenchyma, because embolisms occurred as patches around resin canals and along the stem radius (Figures 5 and 6). In the advanced stage of pine wilt disease, a drastic increase in the embolized xylem area occurred within a few days, as the embolized patches fused together until the whole xylem was embolized. This coincided with a large decrease in water potential and a marked increase in AE rates during both day and night. At this stage, the threshold water potential for cavitation was similar to that during the early stage, but the rate of AE events was much higher (Figure 11). This phase of explosive embolization can be attributed to the sharp decrease in water potential, i.e., runaway embolism, which can occur over the whole transverse area of the xylem when the water potential is below the air-seeding threshold. Embolization seemed to expand tangentially from previous patches (Figures 5 and 6), which accords with the alignment of bordered pits, through which air seeding occurs, on the lateral faces of the tracheids. In the advanced stage of the disease, transpiration rate is negligible because of stomatal closure (Fukuda et al. 1992), however, stomatal closure occurred too late to avoid runaway embolism, because the threshold water potential for cavitation was higher than the threshold for stomatal closure. In conclusion, we successfully monitored cavitation events



advanced stage of the disease. The abrupt decreases in water potential and stem thickness indicated a drastic change in the water status of the whole seedling. The decrease in water potential coincided with both the rapid enlargement of embolized areas and a high rate of AE events. The cumulative AE events in both stem positions were closely correlated with the area of xylem embolized (Figure 10). The increase in the rate of AE events in the current-year shoot preceded that at the stem base. The embolism observed in the middle portion of the stem, between the two AE sensors, showed that the timing of cavitation differed between the stem positions. These results indicate that the AE events corresponded to cavitation in the xylem at each stem position and that cavitation occurred first in the current shoot, then in the MRI-monitored portion, and finally at the stem base. Because the distributions of nematodes and cell disintegration in the advanced stage showed no vertical gradient in small seedlings (Fukuda et al. 1992), the downward development of cavitation cannot be attributed to nematode distribution. Rather, it corresponded with a lower water potential in the upper part of the stem and with higher vulnerability of current-year shoots to cavitation compared with older stems, as suggested by the hydraulic architecture model (Tyree and Sperry 1988). After intensive AEs and embolism, stem thickness showed some recovery after September 5 because leaf wilting and embolism in the upper part of the stem resulted in the release of water tension in the xylem near the strain gauge.


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by measuring AEs caused by pine wilt disease and related the rate of AE events to the occurrence of xylem embolism as observed by nondestructive MR microscopy. The patchy embolism observed in the early stage of pine wilt disease is thought to differ from the phenomenon of cavitation caused by water stress in healthy or latent-stage wilt disease infected pines. The drastic enlargement of the relative area of embolized xylem in the advanced stage of the disease is thought to reflect runaway embolism.

Acknowledgments We thank Dr. Tomoyuki Haishi at MRTechnology Inc., Japan, and Prof. Katsumi Kose at the University of Tsukuba, Japan, for support in constructing and testing the MRI apparatus. We also thank Drs. Takeshi Abe, Tomoko Matsubasa and Sachiko Mori, TOKYO-GAS Frontier Institute, for help in monitoring relative stem diameter.

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Figure 11. Relationship between relative stem diameter (strain gauge (SG) measuring circuit output) and acoustic emission (AE) events in Seedling 2 inoculated with pinewood nematodes on August 12. Latent infection stage, August 11–19; Early stage of disease manifestation: August 20–30; and Advanced stage: August 31–September 7. Arrows indicate threshold water potential (stem diameter) for cavitation.

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