Trees (2000) 14:305–311
© Springer-Verlag 2000
O R I G I N A L A RT I C L E
Atsushi Kume · Naoko Tsuboi · Takami Satomura Masayo Suzuki · Masaaki Chiwa · Kaneyuki Nakane Naoki Sakurai · Takao Horikoshi · Hiroshi Sakugawa
Physiological characteristics of Japanese red pine, Pinus densiflora Sieb. et Zucc., in declined forests at Mt. Gokurakuji in Hiroshima Prefecture, Japan Received: 16 December 1998 / Accepted: 7 January 2000
Abstract The decline of Japanese red pine trees (Pinus densiflora Sieb. et Zucc.) at Mt. Gokurakuji (693 m a.s.l.), 30 km west of Hiroshima city, west Japan, was studied. The effects of air pollution and acid deposition on the physiological characteristics of the trees, especially those of the needles, were investigated. Ozone concentration was not correlated with the physiological status of the needles and SO2 concentration was not high in the declined area. NO2 concentration correlated negatively with needle longevity while it correlated positively with ethylene emission from 1-year-old needles. Average needle longevity was about 2.8 years in non-declined areas; however the longevity was 1.3 years in the most polluted area. The minimal fluorescence at night (F0) of 1-year-old needles decreased with increasing NO2 concentration. The maximum stomatal conductance (gl), net photosynthesis (Pn) and intercellular CO2 concentration (Ci) in the declined areas were lower than in the nondeclined areas (about 50%, 30% and 20% lower, respectively). The lower Ci suggested that the major part of the decrease in Pn can be explained by stomatal restriction. The soil pH, N content and C/N ratio showed no significant difference between the declined and non-declined areas. The physiological disorders of needles were due to the damage by air pollutants, and important roles of NO2 are suggested. Lowering of Pn and the shortening of needle longevity appear to be the main causes of the decline in pines in the forest decline area. A. Kume (✉) The Center for Forest Decline Studies, CREST, Japan Science and Technology Corporation, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan e-mail:
[email protected] Tel.: +81-824-246510, Fax: +81-824-246510 N. Tsuboi · T. Satomura · M. Suzuki · ·M. Chiwa Graduated School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan K. Nakane · N. Sakurai · T. Horikoshi · H. Sakugawa Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan
Key words Air pollution · Ethylene · Chlorophyll fluorescence · Photosynthesis · Pinus densiflora
Introduction Japanese red pine (Pinus densiflora Sieb. et Zucc.) forests in the Seto Inland Sea area of Japan have been severely damaged since the 1970s and approximately onequarter of the pine trees had perished by 1994 (White paper published by Hiroshima Prefecture). The pine forests at Mt. Gokurakuji in the Seto Inland Sea area have also been severely damaged over the last decade. Air pollution, acid rain, infection by the pine wood nematode, and progress of secondary succession have been suggested as causes of the pine tree decline. Some of these factors work simultaneously, but it is not clear how each factor is related to the blight damage. Naemura et al. (1997) reported that the atmospheric NO2 concentration on the seaward side of Mt. Gokurakuji was significantly higher than that on the inland side. They found a high correlation between the mean atmospheric NO2 concentration and the mortality of P. densiflora and Prunus species, the dominant tree layer species. If air pollution and/or acid deposition had accelerated the forest decline, it would be likely that the needles which had been directly exposed to air pollutants would show physiological disorders (Heath 1994). Therefore, diagnosis of the physiological status of the pines is important to elucidate the cause of their decline. In order to investigate the physiological status of the pine needles, we compared (1) stomatal conductance (gl), (2) net photosynthesis (Pn), (3) the electron transfer process in light reactions (PS II), (4) ethylene emission, and (5) needle longevity. Darrall (1989) showed that gl and Pn are sensitive to low amounts of atmospheric pollution. PS II, which is the initial stage of electron flow in photosynthesis, is an important part of photosynthetic activity (Schreiber et al. 1995a). PS II is the most sensitive to environmental stress, especially excess light. Godde and Buchwald
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Fig. 1 Location of study sites on Mt. Gokurakuji, Hiroshima Prefecture, western Japan. Urban areas extended south-east from the base of the mountain. The Sanyo Express Highway and Route 2 are arterial roads in Hiroshima Prefecture. The part of the Sanyo Express Highway shown in the figure opened in 1987
(1992) suggested that the primary light inactivation of PS II was the most likely cause of light-dependent bleaching and the decrease in photosynthetic activity caused by air pollutants. As a plant hormone, ethylene affects various physiological processes and is produced in response to many environmental stresses. The emission of stress ethylene is known to be formed well before visible injuries occur due to the short-term stress of air pollutants (Kimmerer and Kozlowski 1982; Glick et al. 1995), and is used as an indicator for the early diagnosis of forest decline (Fuhrer 1985; Wolfenden et al. 1988). The emission of ethylene is often associated with a decrease of gl (Heath 1996) and the senescence of leaves (Mattoo and Aharoni 1988; Picton et al. 1993). Thus, the aforementioned variables (1–5) may be related to each other. Another important point is that these variables are affected not only by air pollution but also by the stand density of trees, root vigor and soil conditions, such as soil pH, N content and water availability. Therefore, the effects of these factors on physiological characteristics of the pine trees at each site are discussed. Mt. Gokurakuji is located 30 km west of Hiroshima city (34° 23′N, 132° 19′E, 693 m a.s.l., Fig. 1). A few
kilometers to the south of this mountain (on the seaward side) lie the industrial areas and heavy traffic (mainly the Sanyo-Express Highway) of the Hiroshima Bay Area. Obvious damage to the pine forests was observed on the seaward side of this mountain, and little damage was observed on the inland side (Naemura et al. 1997). We surveyed secondary forests with an overstory dominated by P. densiflora trees, about 12 m in height. The understories of these forests are occupied by broad-leaved shrubs, including Eurya japonica Thunberg, Rhododendron reticulatum D. Don and Rhus trichocarpa Miquel. The soil type of each site is acidic brown soil that originated from granite. Annual mean temperature from 1996 to 1997 on the seaward side of Mt. Gokurakuji (120 m a.s.l.) was 16.5°C and annual total precipitation was 1600 mm. Soil pH, N concentrations and C/N ratios were low, but no meaningful differences were detected between the declined areas and the non-declined areas (Table 1). The basal area of stand trees was decreased in the declined areas because of blight damage. However, the ratio of pine to other trees and the fine root biomass per basal area showed no significant trends between the declined and non-declined areas. Atmospheric NO2 concentration on the seaward side of the mountain was significantly higher than that on the inland side, especially at low altitudes (Naemura et al. 1996, 1997; Table 1). The atmospheric NO2 was concentrated at the lower altitude because of the climatic inversion layer that occurred daily on Mt. Gokurakuji below 300 m altitude (Naemura et al. 1996). The atmospheric SO2 was also concentrated at the lower altitude, while the degree of concentration was much less than that of the NO2. The O3 concentration tended to be increase with increasing altitude. From 1996 to 1997, gaseous pollutants (O3, NOx and SO2), meteorological factors (temperature, humidity, and wind direction and speed), rain water and dew were monitored once a month for a period of 24 h around Mt. Gokurakuji (Chiwa et al. unpublished work). In these studies, O3 was measured by UV absorption, NOx was measured by chemiluminescence, and SO2 was measured by fluorescence. Of the air pollutants (O3, NOx and SO2) and ions (Cl–, NO3–, SO42–, Na+, Mg2+, Ca2+, NH4+, HCOO–, CH3COO–, C2O42–), only the air pollutant NOx had a distinctively higher concentration on the seaward side than on the inland side. During the monthly 24-h observations around Mt. Gokurakuji in 1996 and 1997, the peak values of NO and NO2 were 111.0 nl l–1 and 71.8 nl l–1 at 120 m a.s.l. on the seaward side on 28 May 1997, while those on the inland side were 12.1 nl l–1 and 29.8 nl l–1 at 440 m a.s.l. on the same day.
Materials and methods Pinus densiflora is an evergreen coniferous tree, widely distributed in Japan. New needles expand from June to July, and old needles usually fall from September to November. In Hiroshima Pre-
307 fecture, P. densiflora had been widely planted and pure P. densiflora forests are mostly secondary (Miyawaki 1984). Specimens of P. densiflora were sampled from the seaward side (the declined area; 120 m, 280 m, 350 m and 480 m a.s.l. referred to as D120, D280, D350 and D480) and from the inland side (the non-declined area; 440 m, 540 m and 670 m a.s.l. referred to as N440, N540 and N670) of Mt. Gokurakuji in 1997. All seven sites are located on the south slope. Typical symptoms of the infection of pine wood nematode, sudden needle color change and tree death (Kishi 1995), had not been observed for all the trees used in our studies until September 1999. When all the sampled trees were cored and the growth ring observed in September 1998, there were no sudden decreases in the ring widths at least from 1978 to 1998. Therefore, all the pine trees studied had not been infected by pine wood nematode at least until 1998. We also surveyed pines growing in the green-area (nondeclined stand; N210) and those near the heavy traffic intersection (declined stand; D210) on the Hiroshima University campus (34° 24′N, 132° 44′E, 210 m a.s.l.) in 1998. The peak NOx values at D210 were frequently over 200 nl l–1, while those at N210 were at most 40 nl l–1. Fluorescence analysis and photosynthesis measurement At each site three 30- to 50-year-old trees were selected for physiological measurements. These trees were not covered by other trees and seemed to represent each population. The branches located in the top of the canopy surface layer were collected with a long branch cutter, about 12 m in length. The minimal fluorescence (F0) and the photochemical efficiencies of PSII in the dark (Fv/Fm), and in the light (∆F/Fm’) were measured by a photosystem yield analyzer (MINI-PAM and Leaf-clip holder 2030B, Heinz Walz). F0 is indicative of open reaction centers of PSII due to a fully oxidized state of the primary electron acceptor (Schreiber et al. 1995a). Fv/Fm indicate the potential maximal PSII quantum yield (Björkman 1987) and the value of healthy leaves is about 0.8 (Björkman and Demmig 1987). ∆F/Fm’ closely reflects the effective quantum yield of PSII (Genty et al. 1989). Needles were arranged compactly in a parallel array and were clamped with the leaf-clip holder. At night, from 2200 to 0200 hours in May (10-month-old needles) and August (l-month-old needles), Fv/Fm and F0 were measured at each site, when all reaction centers were open (Krause and Weis 1991). On sunny mornings, from 0700 to 1000 hours in May and September, ∆F/Fm’, Pn, gl and intercellular CO2 concentration (Ci) were measured at each site. The canopy surface was exposed to direct sunlight at all sampling times. Pn, gl, Ci and fluorescence were measured within 5 min after cutting the branches. Based on preliminary experiments, Pn, gl, Ci, Fv/Fm, F0 and ∆F/Fm’ were not significantly changed within this time. After exposure for 1 min to 1000 µmol m–2 s–1 of photon flux density (PFD) using an external halogen lamp (2050B, Heinz Walz), ∆F/Fm’ at 1000 µmol m–2 s–1 PFD was measured by the MINI-PAM. Each parameter of fluorescence was calculated according to Schreiber et al. (1995b). At the same time, using different needles of the same twig, Pn, gl and Ci at 1000 µmol m–2 s–1 PFD at a needle temperature of 20°C were measured with an open-flow infrared gas analyzer with a light and temperature control system (LI-6400, LI-COR). The range of ambient CO2 concentration was between 350 µl l–1 and 380 µl l–1 and relative humidity was between 40% and 60%. On each day that measurements were made, and at each site, the soil water potential at 0.2 m and 0.5 m depth was measured by a soil moisture probe (2900FI, Soil Moisture Equipment). The width and length of needles were also measured with a digital caliper (CD-15, Mitutoyo). We used half of the surface area of the needles as the leaf area. In May, current (10-month-old) needles were measured at each site, except at D480, D210 and N210. In September, current (2-month-old) needles and 1-year-old needles (actually 14 months old) were measured at all sites.
Measurement of ethylene emission from needles On sunny mornings in July and September, ethylene emission was measured for aged needles at each study site. The branches were collected in the same way as they were for the photosynthesis measurements. Twenty current or 1-year-old needles were selected from each branch. The needles were enclosed in a glass test tube (34 ml capacity) capped with a double silicon cap. At the same time, ambient air was also sampled as a control. The tube was kept in a temperature-insulated plastic box in the dark and the temperature was kept between 15°C and 20°C for 3 h. Then the gas in the tube was transferred to an evacuated glass vial (5 ml) with a double-ended needle through the Butyl septum. The gas sample (1 ml) was introduced into a gas chromatograph equipped with a glass column (i.d. 3 mm×1.5 m) packed with activated aluminum. The column temperature was 80°C. Ethylene was detected by a flame ionization detector (GC-14B, Shimadzu) and recorded by an integrator (C-R6A, Shimadzu). Needle longevity The average needle longevity was calculated based on 18 shoots collected from the top of the canopy surface layer of six trees in September. The percentage of needles that remained was recorded, and the longevity of the needles was calculated as follows: Needle longevity=Σ[(% of remaining n-month-old needles) n 12–1] where n is the needle age. Statistics Data are presented as mean ± one standard deviation (SD). Scheffe’s multiple range test (Zar 1996) was applied for the analysis of variance (P
