Monitoring of xylem embolism and dysfunction by the acoustic emission technique in Pinus thunbergii inoculated with the pine wood nematode Bursaphelenchus xylophilus

Xylem dysfunction progresses rapidly in Pinus thunbergii infected with pine wilt disease. The present report deals with the timing and process of the extensive dehydration of tracheids by embolism and the subsequent desiccation of the xylem with disease development. An ultrasonic acoustic emission (AE) technique was used to detect embolisms in the xylem of pine trunks. In most of the P. thunbergii saplings inoculated with the pathogen Bursaphelenchus xylophilus, the AE frequency suddenly increased in the second week after inoculation. The high-frequency AE continued for about 3 days and into the nights. Harvesting of specimens at this time revealed that white air-filled patches, representing the dehydrated and dysfunctional areas, had just emerged in the sapwood. The AE events in the night must be due to something other than embolisms in healthy trees. Frequent embolism of tracheids, which was suggested by the elevation of the AE frequency, might occur due to the decrease in the tensile strength of xylem sap. This hypothesis is supported by previously reported data. Host cells that had reacted to infection with B. xylophilus produce and release chemicals which can lower the surface tension of xylem sap. During the second increase of AEs, most of which occurred in the third week, xylem desiccation and needle yellowing progressed. Needle fading then became distinct, and the tree was close to death when the AE frequency dropped during the fourth week. By monitoring the AE, the first physiological abnormality that took place very early after infection was detected.     

Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

树干注药技术预防松材线虫病的效果评价

松材线虫病是松林的毁灭性病害,过去几十年已在日本、韩国及中国等东亚国家迅速传播扩散.加强对非疫区的监测与保护是目前关注的重点,利用1％阿维菌素微囊悬浮剂对马尾松进行树干注药以预防松材线虫病,研究结果表明,树干注药处理区的枯死松树要显著少于不实施注干的对照区,且处理区内枯死松树比率不超过2％.因此,通过树干注药技术施用1％阿维菌素微囊悬浮剂对预防松材线虫病能起到显著效果.     

A rapid staining‐assisted wood sampling method for PCR‐based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR‐based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining‐assisted wood sampling method aiding PCR‐based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5‐mg wood cross‐sections, of 0.5 × 0.5 × 0.015 cm dimension, that were cut from a wood sample of 0.5 × 0.5 × 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5‐mg wood cross‐sections (that adjoined the three 5‐mg wood cross‐sections, mentioned above) were cut. Nematode‐staining‐spots (NSSs) in each of the two stained sections were counted under a microscope at 100× magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus– specific amplicon of 403 bp ( DQ855275 ) was generated by PCR assay from 100.00% of 5‐mg wood cross‐sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx‐detection methods such as PCR assay.     

Direct PCR‐based method for detecting Bursaphelenchus xylophilus,the pine wood nematode in wood tissue of Pinus massoniana

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, leads to serious losses to pine forestry around the world. Pinus massoniana, which is vulnerable to be attacked by the PWN, is the dominant species used in pine forestry in China. The objective of this study is to develop a direct PCR‐based method for detecting B. xylophilus in the wood of P. massoniana without a separate nematode extraction step. A simple procedure was first developed for isolating B. xylophilus DNA in 5 mg pine wood tissue samples harbouring PWN for detection by PCR amplification. A B. xylophilus‐specific amplicon of 403 bp (DQ855275) was generated by PCR from the infested wood tissue. The entire procedure can be completed within 5 h with one pair of primers. This assay can serve as a rapid, cheap and environmentally friendly method to detect B. xylophilus in samples of P. massoniana.     

Long‐term survival and non‐vector spread of the pinewood nematode,Bursaphelenchus xylophilus,via wood chips

The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is transmitted to new host trees by beetles of the genus Monochamus. The increasing interest in imported wood chips from North America for paper production and energy purposes and the corresponding phytosanitary risk of non‐vector transmission of B. xylophilus has been discussed since 1984, the year of the first interception of B. xylophilus in wood chips in the European Union. The long‐term survival of B. xylophilus in wood chips and its non‐vector spread from infested wood chips to non‐infested trees were studied. Pinus sylvestris logs were inoculated with a suspension of B. xylophilus to produce infested wood chips. During the long‐term storage test, B. xylophilus in P. sylvestris wood chips were examined. Four variants, including sealed and openly stored wood chips at both 15°C and 25°C, were studied. For the test of non‐vector spread, B. xylophilus ‐infested wood chips were placed on three‐ to four‐year‐old P. sylvestris saplings under different conditions. Bursaphelenchus xylophilus survived for more than 1 year at both temperatures in the sealed wood chips, which was significantly longer than for the openly stored variant at 25°C. Temperature, tree condition and wood chip location all influenced non‐vector spread through wood chips. Of the 480 trees that were in contact with infested wood chips and showed clear symptoms of pine wilt disease, B. xylophilus were extracted from 42 pines at 25°C and one pine at 15°C. The highest B. xylophilus infestation rates resulting in clear pine wilt disease symptoms (75%) were found in infested wood chips directly attached to stem‐wounded trees at 25°C. However, more variants exhibited B. xylophilus infestation at this temperature; trees with stem or root injuries plus direct contact with infested wood chips to the wounded part were primarily affected. Moreover, non‐vector spread was also detected in stem‐ and root‐injured pines without any direct contact with infested wood chips. Our results confirmed that B. xylophilus can survive for long periods in wood chips and can be transmitted from infested wood chips to damaged trees, but the likelihood of such PWN establishment should be low compared to spread through vectors. These findings must be considered in the pest risk analysis of B. xylophilus, and studies using outdoor trials should be carried out to complete this pest risk analysis.     

Retardation of pine wilt disease symptom development in Japanese black pine seedlings exposed to simulated acid rain and inoculated withBursaphelenchus xylophilus

Three- or 4-year-old Japanese black pine seedlings were exposed to simulated acid rain (SAR) at pH 3 for two months, then inoculated with a virulent isolate (S 10) of pinewood nematodes. The experiments were repeated three times in 1996–1998. The exposure to SAR killed no seedlings, and retarded the development of disease symptoms in the seedlings inoculated with nematodes. In the experiments in 1996 and 1997, however, cessation of resin exudation, and primary decrease in the xylem conductivity occurred earlier in the seedlings exposed to SAR than in those exposed to tap water as a control. These results imply that acid rain at pH 3 influenced an increase in some resistance of Japanese black pine seedlings to pinewood nematode, and that this improved resistance could potentially overcome damage caused by acid rain. The exposure to SAR did not have any significant effect on the water relations of the seedlings itself, suggesting that retardation of the symptom development after the exposure to SAR could not be attributed to the improvement in the water relations. This work was supported in part by a Grants-in-Aid for Scientific Research from the Japan Forest Technical Association (JAFTA).     

Effects of inoculum density of pinewood nematode on the development of pine wilt disease in Japanese black pine seedlings pretreated with simulated acid rain

Six‐month‐old Japanese black pine seedlings (Pinus thunbergii) were exposed to simulated acid rain (SAR) at pH 3 and 2 three times a week. After treatment for 2 months, the seedlings were inoculated with a virulent isolate (S10) of the pinewood nematode (Bursaphelenchusxylophilus), at three inoculum levels (P_i = 50, 160 or 500 nematodes per seedling). In seedlings inoculated with 500 nematodes, both population growth of nematodes and disease development were accelerated by pretreatment with SAR at pH 3 or 2. In seedlings inoculated with 50 nematodes, population growth of the nematodes was suppressed and more time was needed for seedlings to die when pretreated with pH 3 SAR. This suggests that exposure to pH 3 SAR increased not only the progress of mortality, but also simultaneously enhanced the tolerance limit of the seedlings to the pinewood nematode – the critical value of physiological burden (represented as a product of time and initial nematode population) necessary to kill a seedling. Exposure to pH 2 SAR accelerated nematode reproduction in seedlings and increased seedling mortality irrespective of the number of nematodes inoculated.     

Wind profiles in a coastal forest of Japanese black pine (Pinus thunbergii Parl.) with different thinning intensities

Simultaneous measurements of windspeed were made outside and inside a pine (Pinus thunbergii Parl.) coastal forest with different thinning intensities. Synchronously, optical stratification porosity (OSP), which is defined as vertical distribution of the proportion of sky hemisphere not obscured by tree elements inside a forest stand, was estimated using hemispherical silhouettes in each treatment area. Based on the observations, the frequency distribution of velocity, turbulence intensity, wind profile, and the corresponding relationships of these respective parameters with the vertical forest structure (OSP) were examined and compared among the treatment areas. A normal distribution of wind velocity was observed at the lower zone of the forest stand in all treatment areas. The turbulence intensity, the highest value of which was found near the canopy, changed greatly with height, wind velocity, and treatment, and decreased with windspeed and increased with the thinning intensity. It was found that the exponential relationship between windspeed and height could be used to describe the wind profiles within the canopy of the coastal forest. The results demonstrate that the attenuation coefficient of the wind profile corresponds to the grade of thinning intensities. The wind patterns in a coastal forest with different thinning intensities are related to the vertical forest structure, particularly, wind profiles within the canopy are closely correlated with the distribution of OSP. The results indicate that wind profiles can be estimated simply based on the measurement of OSP with a very high coefficient of determination. Reciprocally, the estimation of OSP can also be obtained from the measurement of wind profile. Portions of this paper were presented at the 111st Annual Meeting of the Japanese Forestry Society. Appreciation is due to Professor Masashi Yamamoto, Mr. Kenji Sakioka, Mr. Takahiro Yoshida, Mr. Hirotaka Yamazaki, Mr. Yasunori Hasegawa and the members of the Sabo Division of the Faculty of Agriculture of Niigata University for their help in collecting the field data and in constructing the observation towers.     

Promotion of the population growth of pinewood nematode in 4-month-old Japanese black pine seedlings by pretreatment with simulated acid rain

Simulated acid rain (SAR) at three pH levels (pH 4, 3, 2) was applied to only the top or both the top and roots of 4-month-old Japanese black pine seedlings repeatedly for two months. Then the seedlings were inoculated with a virulent isolate (S10) of pinewood nematode,Bursaphelenchus xylophilus. The exposure to SAR at any level did not kill the seedlings. When inoculated with nematodes, however, the nematode population build-up was significantly higher in the seedlings pretreated with SAR at any level than in the control at the 7th and 17th day after inoculation, and subsequent disease symptom development was also significantly accelerated by the exposure to SAR at pH 2 and 3. This result indicates that even acid rain at pH 4 has the potential for promoting population growth of pinewood nematodes in 4-month-old Japanese black pine seedlings.     

Discoloured and non‐conductive sapwood among six Fagaceae species inoculated with Raffaelea quercivora

Mass mortality of oak trees has been occurring in Japan since the late 1980s. The fungus Raffaelea quercivora has been frequently isolated from discoloured sapwood in dead or wilting trees and inoculation experiments have shown it to be capable of causing wilting and xylem discoloration in several oak species, notably Quercus crispula and Q. serrata. In this study, we inoculated seedlings of six Fagaceae species with R. quercivora and, after 56 days, measured the vertical length of the discoloration and the areas of discoloured and non‐conducting sapwood on stem cross‐sections. The sapwood discoloration and the water non‐conduction areas were larger in Q. crispula and Q. serrata than in the other species.     

Differential spread of discoloured and non‐conductive sapwood among four Fagaceae species inoculated with Raffaelea quercivora

To reveal the relationship between the susceptibility of Fagaceae species to Raffaelea quercivora Kubono et Shin‐Ito and the tangential expansion of regions of discoloured and non‐conductive sapwood among the species, we inoculated branches of three Quercus species and one Castanopsis species with the fungus. The sapwood around the inoculation hole in all four species became non‐conductive in response to the infection before the discolouration. The expansion of the region of non‐conductive sapwood ceased within 2 weeks after the inoculation. The region of non‐conductive sapwood in Q. crispula and Q. serrata was larger than that in Q. glauca and C. cuspidata var. sieboldii. These results suggest that the region of non‐conductive sapwood expanded soon after the infection by R. quercivora. It was also clear that the transverse non‐conductive sapwood has close relationship with the susceptibility among Fagaceae species to R. quercivora.     

杨树溃疡病及松材线虫病对树体干部电指标的影响

对杨树溃疡病及松材线虫病对树木电指标的影响进行研究.结果表明:杨树溃疡病胁迫下,毛白杨扦插苗POD酶活性先增大后减小,PPO酶活性持续增大,树木干部电容和单位电容呈减小趋势,而树木干部阻抗呈增大趋势,最先表现出统计学差异性的指标是POD酶活性,其次为树木干部电容,之后为树体干部阻抗,最后是PPO酶活性;树木干部电容和单位电容与马尾松枯针率均有显著的正相关关系,同时利用树体干部电容0.5 nF为阈值,判断马尾松是否感染松材线虫的准确率可达89.26%.     

A key effector,BxSapB2, plays a role in the pathogenicity of the pine wood nematode Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses in pine forests. The plant‐parasitic nematodes have a complex life cycle that includes the secretion of effector proteins through a stylet into the host cell to promote parasitism. In this study, SignalP 4.1 and TMHMM 2.0 were used in preliminary screens for candidate effectors and were expressed in Nicotiana benthamiana through the PVX virus expression vector. The yeast signal sequence trap system was used to further study the function of the signal peptide of an effector, BxSapB2. In situ hybridization was conducted to investigate the localization of BxSapB2, followed by RNA interference technology (RNAi) to assess the functions of BxSapB2. The results demonstrate that BxSapB2 is a secreted protein that induces cell death in N. benthamiana and is highly expressed in esophageal gland cells and amphids of B. xylophilus. BxSapB2 was determined to be related to the pathogenicity of B. xylophilus. The results of this work indicate that BxSapB2 plays an important role in the interactions between B. xylophilus and the hosts.     

松材线虫携带的一株致病细菌对非寄主植物的毒性及对黑松LOX酶活性的影响

在实验中发现松材线虫携带的一株致病菌株荧光假单孢菌Pseudomonas fluorescens GcM51A毒素液对玉米、小麦、木耳菜和油麦菜均有毒害作用。以松针为材料,用不同浓度的毒素液处理,测定不同时间内松针细胞脂氧和酶活性的变化。结果表明毒素原液处理的松针LOX活性在处理后的18h达到高峰,随后逐渐下降;稀释5倍处理的松针LOX活性在6～12h呈上升趋势,之后逐渐下降。     

Responses of water-stressedPinus thunbergii to inoculation with avirulent pine wood nematode (Bursaphelenchus xylophilus): Water relations and xylem histology

The effect of water-stress conditioning on water relations and histological features ofPinus thunbergii Parl. inoculated with avirulent isolate ofBursaphelenchus xylophilus (Steiner and Buhrer) Nickle, pine wood nematode, were investigated. Pines were kept under 8 days cycle of severe water stress. One-half of the water-stressed pines died as a result of infection by avirulent pine wood nematode and water stress tended to induce increased susceptibility and/or decreased resistance of pines to avirulent pine wood nematode. In dead pines, the water conducting function of xylem was lost, and all of the parenchyma cells died. In surviving pines, the xylem hydraulic conductivity and the xylem water content were significantly reduced (12 to 23% and 77 to 83%, respectively) compared to controls. Safranin dye perfusion of excised axis stem segments indicated that the water conductance was limited to the very narrow peripheral area of xylem. Embolism caused by cavitation in the tracheids occurred in the central part of xylem and in that dysfunctional region of the xylem the axial parenchyma cells surrounding the epithelial cells, and ray parenchyma cells partly degenerated but the epithelial cells survived. The disruption of tracheid shape observed in surviving pines indicates that avirulent pine wood nematode temporarily disturbed cell division of the cambium. Considering the differences in responses between dead pines and surviving pines after inoculation with avirulent pine wood nematode, the death of water-stressed pines apparently resulted from death of cells, in particular the vascular cambium and the loss of xylem hydraulic function by cavitation.     

Evaluation of intra-ring wood density profiles using NIRS: comparison with the X-ray method

Key message

Pith-to-bark wood density profiling is interesting in forestry science. By comparing it with the X-ray method, this study proved that a fiber optic NIR spectrometer with a high-precision displacement system could accurately measure intra-ring wood density with a spatial resolution of 0.5 mm.

Context

Most near-infrared spectroscopy (NIRS) studies for wood density determination use samples that have been pulverized beforehand. Attenuation of ionizing radiation is still the standard method to determine wood density with high spatial resolution. However, there is evidence that NIRS could be an accurate and affordable method for determining intra-ring density in solid wood strips.

Aims

In this study, we research whether the results published for intra-ring density predictions in wood can be improved when calibrated with X-ray microdensitometry.

Methods

The measurements were made using a fiber optic probe with a separation between measurement points of 0.508 mm in a range between 1200 and 2200 nm. A total of 4520 density points were used to create partial least squares regression (PLSR). X-ray densitometry data were used as reference values. Twenty PLSR calibrations were randomly executed on 31 samples collected from 28 Pinus radiata D. Don trees.

Results

Upon selecting 20 latent variables, the R ² value was 0.873 for the training group and 0.895 for the validation group, while RMSEP values are 43.1 × 10^?3 and 47.1 × 10^?3 g cm^?3 for the training and validation groups, respectively. The range error ratio (RER) was 13.7.

Conclusion

The RER was high and almost in the range suggested for quantification purposes. Results are superior to wood density studies in the literature which do not employ spatial resolution and to those found in studies using hyperspectral imaging.

     

Localized within‐ and between‐tree variation in nematode distribution during latent state of pine wilt disease makes the disease status cryptic

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, an epidemic disease that has severely damaged pine forests in East Asia. The disease has spread to northern areas in Asia and parts of Europe. To prevent disease spread as the forefront of damage prevention, a better understanding of infection status is highly important. Not all infected trees show disease symptoms, and such asymptomatic PWN‐carrying trees are likely to be overlooked and can become a pathogen reservoir. To elucidate PWN infection status in asymptomatic trees, we performed PWN inspection of branches and trunks in 21 test trees in two different conditions: trees that had experienced PWN inoculation and those with suspected PWN infection that had experienced transient foliage discoloration. We detected PWNs in eight test trees (38%) and in 13 (1.5%) of a total of 843 samples. The difference in these percentages suggests that nematode inhabitation was highly localized within the trees, possibly owing to the restricted migration of PWNs. Our data demonstrated that trees that were once weakened but recovered their vigour can persist, as the asymptomatic carriers, in the forest. The implications for disease control are also discussed.