Pinus Densiflora Seedlings Research Articles

Two-year survival of Tricholoma matsutake ectomycorrhizas on Pinus densiflora seedlings after outplanting to a pine forest

Two-year survival of Tricholoma matsutake ectomycorrhizas on Pinus densiflora seedlings after outplanting to a pine forest

In vitro ectomycorrhization of Tricholoma matsutake strains is differentially affected by soil type

The Japanese delicacy Tricholoma matsutake has been conducted in vitro ectomycorrhizal syntheses for more than 20 y. The development of its ectomycorrhizal structures varies among experimental systems. Here, we examined the effects of soil-fungus interactions on the early stage of in vitro T. matsutake ectomycorrhization. Axenic Pinus densiflora seedlings were transplanted into autoclaved natural inorganic soil, inoculated with the cultured mycelium of T. matsutake, and incubated for 90 d in vitro. Both soil type and fungal strain significantly affected host plant growth; host plant growth and mycorrhization levels significantly differed among soil type/fungal strain combinations. Therefore, the selection of T. matsutake strains for optimal mycorrhization must take into account such fungal and soil properties.

Growth Characteristics of One-year-old Container Seedlings of Pinus densiflora by Irrigation Level

Growth Characteristics of One-year-old Container Seedlings of Pinus densiflora by Irrigation Level

Soil spore banks of ectomycorrhizal fungi in endangered Japanese Douglas-fir forests

Interactions between trees and ectomycorrhizal fungi are critical to the growth and survival of both partners. However, ectomycorrhizal symbiosis has barely been explored in endangered trees, and no information is available regarding soil spore banks of ectomycorrhizal fungi from forests of threatened trees. Here, we evaluated soil spore banks of ectomycorrhizal fungi from endangered Japanese Douglas-fir (Pseudotsuga japonica) forests using bioassay approaches with congeneric P. menziesii and Pinus densiflora seedlings in combination with molecular identification techniques. Rhizopogon togasawariana was predominant in soil propagule banks and was found in all remaining P. japonica forests when assayed with P. menziesii, while no colonization of this fungus was observed on Pinus seedlings. Given the observed specificity of R. togasawariana for P. menziesii and its phylogenetic position within the Pseudotsuga-specific Rhizopogon lineage, its geographical distribution is likely restricted to the remaining Japanese Douglas-fir forests, indicating a high extinction risk for this fungus as well as its endangered host. Spore banks of R. togasawariana remained highly infective after preservation for 1 year or heat treatment at 70 °C, suggesting an ecological strategy of establishing ectomycorrhizal associations on regenerating Japanese Douglas-fir seedlings after disturbance, as observed in other Rhizopogon–Pinaceae combinations. Therefore, the regeneration of Japanese Douglas-fir seedlings may depend largely on the soil spore banks dominated by R. togasawariana, which has co-evolved with the Japanese Douglas-fir for over 30 million years. More attention must be paid to underground ectomycorrhizal fungi for the conservation of endangered tree species, especially in the era of human-induced mass extinction.

토양개량제 처리가 물푸레나무와 소나무 묘목의 생장과 양분농도에 미치는 영향

토양개량제 처리가 물푸레나무와 소나무 묘목의 생장과 양분농도에 미치는 영향

Effects of Open-field Artificial Warming and Precipitation Manipulation on Physiological Characteristics and Growth of Pinus densiflora Seedlings

Climate change affects plant responses on physiological characteristics and growth, and Pinus densiflora, one of the major tree species in Korea, are expected to be particularly vulnerable to rising temperature and increased precipitation. This study was conducted to investigate the effects of an open-field warming and precipitation manipulation on physiological characteristics and growth of P. densiflora seedlings. Seedlings of 2-year-old P. densiflora were planted in April, 2013, in open-field nursery located at Korea University. The air temperature of warmed plots had been set to be 3℃ higher than the control plots using infrared lamps. Precipitation was manipulated to be 30% lower or higher than the control, using transparent panels and drip irrigation. Net photosynthetic rate, total chlorophyll content, seedling height, root collar diameter and biomass were measured from April, 2014 to April, 2015. The increase in new shoot biomass from warming was statistically significant, with the biomass in warmed plots about 2-fold higher than in the control plots in 2014 and 2015. This result might be related to advanced bud burst and increased occurrence of abnormal new shoots in warmed plots. Meanwhile, the results of net photosynthetic rate, total chlorophyll content, seedling height, root collar diameter and total biomass from warming and precipitation manipulation were not statistically significant, but tendencies of lower net photosynthetic rate and higher seedling height and biomass in warmed plots compared to the control were shown. Such might be speculated as results of the extended growth period. When root to shoot (R/S) ratio was calculated from the biomass data obtained in April 2014 and April 2015, increased R/S ratio was observed regardless of the treatments applied. Drought tolerance of P. densiflora and particularly low annual precipitation observed in 2014 were suggested as the possible reasons.

Short-term effects of warming treatment and precipitation manipulation on the ecophysiological responses of Pinus densiflora seedlings

The aim of this study was to investigate the short-term effects of warming treatment and precipitation manipulation on the growth, photosynthetic rate, and chlorophyll content of Pinus densiflora seedlings based on a climate change scenario in Korea. Two-year-old P. densiflora seedlings were planted in a nursery in April 2013. The air temperature of warmed plots (W) was set to increase by 3 °C compared to control plots (C) using an infrared heater in May 2013. The three precipitation manipulations consisted of decreased precipitation using transparent panels (-30%; P-), increased precipitation using pumps and drip irrigation (+30%; P+), and a control (0%; P0). Root collar diameter and seedling height of P. densiflora were measured in April and October 2013. Net photosynthetic rate and total chlorophyll contents were measured from June to October 2013. Warming treatment increased the growth of root collar diameter, and the interaction effect of the warming treatment and precipitation manipulation on it was also significant. In contrast, no significant effects were found for the growth of seedling height. These results demonstrate that warming treatment might affect the growth of root collar diameter and seedling height differently. It was also found that WP- treatment decreased net photosynthetic rates. This pattern might be due to the soil moisture availability, as soil moisture content was lowest in the WP? treatment and net photosynthetic rate was elevated with increasing soil moisture content. Meanwhile, warming treatment increased the total chlorophyll content. The results suggest that chlorophyll synthesis was promoted by a temperature increase in the leaves. The current study, which simulated a climate change environment in an open field, provides important information for the prediction of the ecophysiological effects of changes in temperature and precipitation on P. densiflora seedlings in Korea.

Comparison of Genetic Variation between Pre-practice Mature Trees and Post-practice One-year Old Seedlings in Pinus densiflora Natural Regeneration Stands

We studied the genetic impact of natural regeneration practices, such as Single seed tree, Group seed tree, Patch clear cutting and Alternate strip clear cutting systems, by comparing the nuclear microsatellite(nSSR) variation of post-practice natural regeneration one-year old seedlings of Pinus densiflora to that of pre-practice mature trees. The levels of genetic diversity of seedlings (A=13.6, =4.3, =0.571, =0.597) were similar to those of mature trees (A=13.4, =4.3, =0.596, =0.598) and the differences in the level of genetic diversity between seedlings and mature trees for each of the practices were not statistically significant. The degree of genetic differentiation between seedlings and mature trees was very low (=0.002) and the pairwise values between seedlings and mature trees for all practices were less than 0.01. Overall, the natural regeneration practices appeared to have only minor impacts on the genetic diversity and the genetic composition in the studied P. densiflora stands. For a better understanding of the genetic effects of natural regeneration practices, subsequent studies such as temporal genetic variation of seedlings formed by crossing among post-practice mature trees should be considered.

한국산 가송이(가칭)의 균사생장 특성과 소나무에 외생균근 형성

Tricholoma bakamatsutake is one of the edible ectomycorrhizal mushrooms as an allied species of Tricholoma matsutake. This is the first report on physical characteristics of T. bakamatsutake strains collected from Quercus mongolica forests in Korea. The pure cultures of these strains were isolated from the tissues of fruit bodies and the culture characteristics were investigated under different conditions (media, temperatures, nitrogen sources). Most strains showed the highest mycelial growth on potato dextrose agar (PDA) at 20 or 25 o C. Two strains of T. bakamatsutake preferred the ammonium-form rather than the nitrate-form as an inorganic nitrogen source. T. bakamatsutake showed significantly slower mycelial growth when compared with T. matsutake from a Korean forest, although the optimum culture conditions for the two allied species were similar. We also tested the ability to form mycorrhizae as well as cellulase activity of T. bakamatsutake. All strains showed cellulase activity on a carboxymethylcellulose (CMC) agar plate. The mycorrhizae on axenic Pinus densiflora seedlings were formed by two strains of T. bakamatsutake after 3 or 8 months of inoculation. P. densiflora seedlings inoculated with T. bakamatsutake had a much higher biomass than un-inoculated seedlings.

기후변화 영향 모의를 위한 실외 실험적 온난화 및 강수 조절 시스템 설계 연구

소나무 묘목을 대상으로 한반도 기후변화 시나리오에 근거한 50년 후 온도 <TEX>$3^{\circ}C$</TEX> 증가와 강수량 30% 변화의 영향을 모의하고자 실외에서 온난화 처리와 강수 조절을 수행할 수 있는 시스템을 설계하였다. 이를 위하여 2013년 4월에 묘포장을 조성하고 2년생 소나무 묘목을 식재한 다음 5월부터 적외선등을 이용하여 온난화 처리구(W)의 대기온도를 대조구(C)에 비하여 <TEX>$3.0^{\circ}C$</TEX> 높게 설정하여 가열하였으며, 강수 대조구(<TEX>$P^0$</TEX>)와 강수 차단 덮개를 이용한 강수 감소(대조구 대비 -30%; <TEX>$P^-$</TEX>) 조절, 그리고 펌프와 점적관수를 통한 강수 증가(대조구 대비 +30%; <TEX>$P^+$</TEX>) 조절을 실행하였다. 온난화 처리구의 대기 온도는 초기에 대조구에 비하여 평균 <TEX>$2.2^{\circ}C$</TEX> 높았으나 이후 점차 목표치에 근접한 <TEX>$3.0^{\circ}C$</TEX>로 유지되었다. 또한 온난화 처리에 따른 평균 토양 온도는 온난화 처리구에서 대조구보다 평균 <TEX>$3.1^{\circ}C$</TEX> 높게 나타났다. 강수 증가 및 감소 조절에 따른 평균 토양 수분 함량은 온난화 처리구에서 <TEX>$P^0W$</TEX>에 비하여 <TEX>$P^+W$</TEX>는 13.9% 증가하고, <TEX>$P^-W$</TEX>는 10.0% 감소하였으며, 온난화 대조구 중에서 <TEX>$P^+C$</TEX>는 23.7% 증가하고, <TEX>$P^-C$</TEX>는 7.6% 감소하였다. 환경요인의 모니터링을 통하여 실외 실험적 온난화와 강수 조절 시스템이 적정하게 설계되고 가동됨을 확인할 수 있었다. The objective of this study was to establish an open-field experimental warming treatment and precipitation manipulation system to simulate climate change impact for Pinus densiflora seedlings based on a climate change scenario in Korea. Two-year-old seedlings were planted in a nursery in April, 2013. The air temperature of warmed plots (W) was set to increase by <TEX>$3.0^{\circ}C$</TEX> compared to control plots (C) using infrared lamps from May, 2013. The three precipitation manipulation consisted of precipitation decrease using transparent panel (-30%; <TEX>$P^-$</TEX>), precipitation increase using pump and drip-irrigation (+30%; <TEX>$P^+$</TEX>) and precipitation control (0%; <TEX>$P^0$</TEX>). Initially, the air temperature was <TEX>$2.2^{\circ}C$</TEX> higher in warmed plots than in control plots and later air temperature was maintained close to the target temperature of <TEX>$3.0^{\circ}C$</TEX>. The average soil temperature was <TEX>$3.1^{\circ}C$</TEX> higher in warmed plots than in control plots. Also the average soil moisture content after the precipitation manipulation increased by 13.9% in <TEX>$P^+W$</TEX> and decreased by 10.0% in <TEX>$P^-W$</TEX> compared to <TEX>$P^0W$</TEX>, and increased by 23.7% in <TEX>$P^+C$</TEX> and decreased by 7.6% in <TEX>$P^-C$</TEX> compared to <TEX>$P^0C$</TEX>. It was confirmed that the open-field experimental warming and precipitation manipulation system was properly designed and operating.

Synthesis of Japanese Boletus edulis ectomycorrhizae with Japanese red pine

Boletus edulis is a well-known ectomycorrhizal mushroom. Although cultivation has been widely attempted, no artificial fruiting has been achieved owing to difficulties associated with mycorrhizal synthesis and acclimatization in fields. We collected fifteen B. edulis basidiomata samples from locations in Japan and identified them microscopically and by phylogenetic analysis of their nuclear ribosomal internal transcribed spacer (ITS) regions. Pure culture isolates of B. edulis were established efficiently on malt extract agar medium, and one isolate, EN-63, was inoculated to axenic Pinus densiflora seedlings in vitro. Brownish ectomycorrhizal tips were observed on the pine lateral roots within four months of inoculation. Ten pine seedlings that formed ectomycorrhizae were acclimatized under laboratory and greenhouse conditions. At four months after transplant, mycorrhizal colonization by B. edulis was observed on newly grown root tips under laboratory conditions, but no B. edulis ectomycorrhiza survived under greenhouse conditions. These results suggest that B. edulis ectomycorrhizae synthesized in vitro with P. densiflora requires additional steps for acclimatization to greenhouse conditions.

Growth characteristics of Pinus densiflora seedlings by root pruning intensity

This study was conducted to examine the effect of root pruning intensity on the seedling growth of Pinus densiflora. One-year-old seedling was pruned by 0%, 25%, 50% and 75% from the total root length before transplanting. After one year, above-ground growth(survival rate, seedling height, root-collar diameter, shoot dry weight, branch number and needle weight) and root growth(root length and root dry weight) were investigated. Survival rate of pruned seedlings was better than that of non-pruned seedlings. However, there was no significant differences among root pruning treatments. Excellent above-ground growth was found in non-pruned and 25% root-pruned seedlings. The seedlings pruned by 25% showed superior root to shoot ratio and seedling quality index, which were usually used as measures to determine seedling quality. Therefore, root pruning was highly recommended before transplanting of 1-0 bare root seedling of P. densiflora for enhancing survival rate. In this case, appropriate root pruning intensity was 25% of the total root length of seedling.

Effects of open-field warming and precipitation manipulation on net photosynthetic rate of Pinus densiflora seedlings

Effects of open-field warming and precipitation manipulation on net photosynthetic rate of Pinus densiflora seedlings

실내식물과 소나무의 오존에 대한 광합성 능력, 항산화 효소의 활성, 해부학적 반응

The purpose of this study was to identify the effects of ozone pollution on the one woody species and two indoor plants in controlled environment. Pinus densiflora, Spathiphyllum patinii and Epipremnum aureum seedlings were exposed in both control and ozone chambers to investigate photosynthetic rate, water use efficiency, antioxidative enzyme activities such as GR(Glutathione reductase) and APX(Ascorbate peroxidase) activity and leaf anatomical response. Ozone was fumigated 8 hours for a day with 30 ppb concentration for 50 days. Pinus densiflora seedlings showed no significant difference on photosynthetic rate, water use efficiency, antioxidant enzyme activity during ozone exposure. Ozone concentration (30 ppb in this study) is not high enough to generate ozone damage on Pinus densiflora species. In contrast, ozone generally altered photosynthetic rate, antioxidant enzyme (especially GR) activity and leaf anatomy in two indoor species (Spathiphyllum patinii and Epipremnum aureum) exposed in ozone chamber were significantly differ from those of control in every measurement. These data suggest that two indoor species(Spathiphyllum patinii and Epipremnum aureum) are more sensitive to ozone than Pinus densiflora.

Effects of Media Formulation on the Growth and Morphology of Ectomycorrhizae and Their Association with Host Plant

Tricholoma matsutake and Rhizopogon roseolus form ectomycorrhizal (ECM) association with their host plant on natural habitats. The main objective of this study was to test mycelial growth, morphology, and host plant survival both in vitro and in vivo when treated with enriched media. Aseptically germinated seedlings of Pinus densiflora and P. thunbergii were inoculated with the strains of T. matsutake and R. roseolus, respectively. Under in vitro conditions mycelial growth rates performed best on pH 5 and were better on Modified-Melin-Norkrans-(MMN) based medium and Potato Dextrose Agar (PDA); addition of micronutrients and vitamins in MMN mycelial growth rates had 6–27% differences. Without ECM, plant survival rates on standard media were 30% to below 30% and by inclusion of elements they were 50% to 80%. On in vivo, soil containing different media with ECM allowed successful mycorrhizal association and increased seedling survival rates approximately 100%. Our findings confirm that MMN and PDA allowed higher mycelial growth but poor plant survival (&lt;30%); however, enriched media supported 100% plant survival with successful ECM associations. The present method is advantageous in terms of giving objectivity for ECM by employing suitable media for strains and host plant, and making it possible for mass production of ECM-infected seedlings.

Growth response of Pinus densiflora seedlings to different fertilizer compound ratios in a recently burned area in the eastern coast of Korea

This study was conducted to determine fertilizer compound ratios suitable for soil conditions and tree seedling growth in recently burned areas in Korean forests. Currently, the conventional fertilizer ratio applied to undisturbed forests in Korea is N:P:K 3:4:1. In this study, Japanese red pine (Pinus densiflora S. et Z.) seedlings planted in the burned area were fertilized over four growing seasons with the following NPK compound ratios: unfertilized (CON), 3:4:1, 6:4:1, 3:8:1, and 3:4:2. Fertilization generally increased current-year needle nutrient concentrations of the seedlings, and the chlorophyll a:b ratio in CON was significantly lower than in all fertilized plots. Fertilization significantly affected the growth of the pine seedlings, which had 71–87 % more height growth and 29–67 % increased root collar diameter (RCD) compared to CON. The increases in height and RCD were significantly higher with the 6:4:1 and 3:8:1 ratios than with the 3:4:1 ratio. The 3:4:2 and 3:4:1 fertilizer ratios had no effect on the RCD growth of seedlings. This suggests that the early growth of pine seedlings could be improved by providing high N and P supplies to areas affected by forest fires rather than the conventional fertilizer ratio of 3:4:1 in Korean forest soils. Therefore, application of the suitable fertilization ratio may be a very effective way to reduce reforestation cost as well as to shorten restoration period.

In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora

Amanita caesareoides is a sister species of Amanita caesarea, also known as Caesar's mushroom and one of the most desirable edible mycorrhizal mushrooms. However, cultivation of Caesar's mushrooms has not yet been successful due to the difficulties involved in establishing pure cultures. In this study, we established pure cultures of four Asian Caesar's mushroom species, i.e., A. caesareoides, Amanita javanica, Amanita esculenta, and Amanita similis, which were identified by sequence analysis of their rDNA internal transcribed spacer (ITS) region. Five selected isolates in A. caesareoides, A. javanica, and A. esculenta were tested for ectomycorrhizal syntheses with axenic Pinus densiflora seedlings in vitro. Ectomycorrhizal tips of each fungal isolate tested were observed on pine lateral roots within 5 months of inoculation. Seventeen pine seedlings that formed ectomycorrhizas in vitro with these three Amanita species were acclimatized under non-sterile conditions. Seven months following acclimatization, ectomycorrhizal colonization by A. caesareoides was observed on newly grown root tips, which was confirmed by polymerase chain reaction restriction fragment length polymorphism analysis of the fungal rDNA ITS region. Two other Amanita species also survived during ectomycorrhizal acclimatization. These results suggest that the cultivation of A. caesareoides and its relatives can be attempted through mycorrhizal synthesis using P. densiflora as a host. This is the first report of in vitro mycorrhization of Asian Caesar's mushrooms and their acclimatization under non-sterile conditions.

Helium Ion Microscopy of Uncoated Pine Leaves

A recently introduced helium ion microscopy (HIM) was employed to observe uncoated pine leaf specimens. Adult leaves were collected from the seedlings of Pinus densiflora and P. rigida, air-dried at room temperature, and observed by HIM without metal coating. Ovoid or round stomata and distinct Florin rings could be discerned. The epicuticular waxes were present in the epistomatal chambers and Florin rings of stomata on the leaf surface. The epicuticular waxes were mostly straight, cylindrical, and ca. 1 <TEX>${\mu}m$</TEX> in length. The epistomatal chambers of P. rigida were filled with the epicuticular waxes, whereas those of P. densiflora were not filled with the epicuticular waxes. Based on their micromorphology, the epicuticular wax structures of the pine species were identified as tubules. These results suggest that the HIM could be used for the investigation of the plant stomata and epicuticular waxes of uncoated plant leaves. Due to the smaller ion probe and interaction volume, the HIM has advantages over conventional field emission scanning electron microscopy in terms of image resolution and charge neutralization.

Effect of Soil Ameliorators on Ectomycorrhizal Fungal Communities that Colonize Seedlings of Pinus densiflora in Abandoned Coal Mine Spoils

In this study, the effect of soil ameliorators on ectomycorrhizal (ECM) fungal communities in coal mine spoils was investigated. Organic fertilizers and slaked lime were applied as soil ameliorators in 3 abandoned coal mine spoils. One year after the initial treatment, roots of Pinus densiflora seedlings were collected and the number of ECM species, colonization rate, and species diversity were assessed. The results showed that the soil ameliorators significantly increased ECM colonization on the roots of P. densiflora. The results suggest that soil ameliorators can have a positive effect on ECM fungi in terms of growth of host plants and show the potential use of soil ameliorator treatment for revegetation with ECM-colonized pine seedlings in the coal mine spoils.

Ectomycorrhizal fungal community associated with naturally regenerating Pinus densiflora Sieb. et Zucc. seedlings on exposed granite slopes along woodland paths

We investigated the ectomycorrhizal (EcM) fungal community of naturally regenerating Pinus densiflora seedlings on exposed decomposed granite (DG) slopes along woodland paths in the Republic of Korea. We sampled three- to five-year-old seedlings from slopes where DG was exposed (habitat S) and from the surrounding forest edge or gaps (habitat F). We classified EcMs into morphological groups and counted the number of root tips. The fungal taxa were determined by sequencing the internal transcribed spacer region of their nuclear rDNA. We observed a total of 27 EcM fungal taxa. Atheliaceae and Rhizopogon were the dominant taxa on exposed DG slopes. The fungal species compositions were similar between habitats (0.59 as calculated by the Morishita–Horn index). Fourteen fungal taxa were common to both habitats, and these accounted for more than 90% of the relative abundance in each habitat. There were no significant differences in relative abundance and colonization frequency of each EcM fungal taxon between the habitats, except for Russula sp. 2, which was significantly more frequent in habitat F than in habitat S. Our results indicated that P. densiflora seedlings on exposed DG slopes associated with EcM fungi that were common in forests and not specific to the exposed DG slopes.