Pathogenicity testing of four Bursaphelenchus species on conifer seedlings under greenhouse conditions

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, J. Agric Res. 48, 1934, 949), Nickle (J. Nematol. 2, 1970, 375), is the causative agent of the pine wilt disease and causes serious damage to pine forests around the world. During a survey for the pinewood nematode, four other Bursaphelenchus species (Bursaphelenchus mucronatus, B. sexdentati, B. anamurius and B. vallesianus) were isolated from wilted pine trees in Turkey. To understand the effects of these Bursaphelenchus species on wilting of pine trees, a study was conducted under greenhouse conditions. Two‐year‐old seedlings of three pine species (Pinus nigra, P. brutia and P. pinea) and one cedar species (Cedrus libani) were used. Fifteen seedlings of each species were inoculated with nematodes and 10 seedlings of each species served as controls. The inoculum densities used for each seedling contained approximately 1000 (±100) nematodes of all life stages in 0.25 ml of distilled water. The first wilting symptoms were observed in the fifth week in all pine species but not in the cedar seedlings. All seeding mortality occurred between the 5th and 13th weeks of the study; no mortality was observed outside of this period. The most pathogenic nematode species was B. mucronatus, closely followed by the other species. The most susceptible seedling species was P. nigra, and C. libani was the most resistant species.     

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.     

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.     

松材线虫病在纯松林内自然扩散规律的研究

在偏僻地带的大面积轻微发病纯松林内通过反复强度清理病死、濒死松树及所有可疑松树，及在中心部位人为设置“中心侵染源”松木段，模拟松材线虫病Ｂｕｒｓａｐｈｅｌｅｎｃｈｕｓｘｙｌｏｐｈｉｌｕｓ自然扩散。经定期定点观察，初步摸清了华东丘陵地带松材线虫病及松褐天牛在纯松林内自然扩散规律，为松材线虫病治理提供了依据     

Spatiotemporal analysis of pine wilt disease: Relationship between pinewood nematode distribution and defence response in Pinus thunbergii seedlings

Pine wilt disease is of major concern as it has destroyed pine forests in East Asia and Europe. Several studies have suggested that invasion by the pinewood nematode (PWN) Bursaphelenchus xylophilus, which causes this disease, evokes an excessive defence response in pine trees, resulting in tree death. However, few studies have quantitatively evaluated the correlation between PWN distribution and tree defence responses. Therefore, the present study aimed to quantify the number of PWNs and expression levels of putative pathogenesis‐related (PR) genes in different positions of Japanese black pine (Pinus thunbergii) seedlings over time. To quantify the number of PWNs in the seedlings, we used TaqMan quantitative real‐time PCR (qPCR) assay. During the early phase of infection, most PWNs were distributed around the inoculated sites, with only a small number being detected at distant sites, but the expression levels of PR genes were highly upregulated throughout the seedlings. Both the number of PWNs and expression levels of PR genes then increased drastically throughout the seedlings, all of which exhibited external symptoms. Thus, it appears that the rapid migration of PWNs induces a defence response throughout the seedling; however, this may not be effective in controlling these parasites, thereby ultimately leading to plant death.     

松材线虫侵害马尾松林后群落谱系多样性和结构动态

调查松材线虫Bursaphelenchus xylophilus侵害马尾松Pinus massoniana林不同时间的4个群落和邻近的1个常绿阔叶林群落,利用群落净谱系亲缘关系指数(NRI)和净最近种间亲缘关系指数(NTI)等,分析松材线虫侵害马尾松林后群落谱系结构和谱系多样性动态。结果表明:松材线虫侵害后,群落物种和谱系多样性下降,侵害12 a后与常绿阔叶林的多样性最接近,侵害4,8 a群落内的物种和谱系与常绿阔叶林相似性最高,侵害0 a的与常绿阔叶林相似性最低;松材线虫侵害0,4,8 a的整个群落的谱系结构发散,侵害12 a与常绿阔叶林的谱系结构聚集。松材线虫侵害后,马尾松林群落向常绿阔叶林演替,同时,环境过滤对整个群落物种的组成起主要筛选作用。     

Inoculation of young pine seedlings with Peridermium pini from north-east Scotland

Seedlings of Pinus sylvestris, P. nigra ssp. laricio, and P. mugo vars mughus, rostrata and pumilio were inoculated at their cotyledon stage with aeciospores of Peridermium pini from north-east Scotland. Infection was determined by the presence of both mycelia and haustoria in free-hand or paraffin sections. In the second growing season, infected seedlings showed symptoms such as stem-swelling, development of spermogonia and aecia, and death. In a second experiment, young P. sylvestris seedlings from seven seed sources collected in Great Britain were inoculated and the infection were examined after 6 weeks. In the samples of 50 seedlings from each source, 30–70% seedlings were infected.     

Pathogenicity of canadian isolates of the Bursaphelenchus xylophilus (pinewood nematode) to provenances of Pinus sylvestris and Pinus contorta as grown in Finland: A greenhouse study

Seedlings of Scots pine (Pinus sylvestris L.) and lodgepole pine (P. contorta Dougl.) provenances, as grown in Finland, were inoculated with “m”; and “r”; “forms”; of the pinewood nematode (Bursaphelenchus xylophilus) from Canada, an m form isolate from France and an r form isolate from Missouri, USA. Scots pine was highly susceptible to an Alberta r form and a British Columbia m form isolate and moderately susceptible to two Quebec m form isolates. Lodgepole pine was higly susceptible to the two r form (Alberta and Missouri) isolates and moderately susceptible to the British Columbia m form and the two Quebec m form isolates. Mortality of both pines after inoculation with the French isolate was inconsistent. Mortality of both pines occurred more rapidly following inoculation with r form than with m form nematodes. Large numbers of nematodes were generally found in the tissues of both pines. Our results with seedlings need to be corroborated by inoculating larger field‐grown trees.     

松材线虫发病条件的研究概况

研究和综述了寄生松科植物的伞滑刃属(Bursaphelenchus)的线虫23种,松材线虫的寄主松科植物108种、携带昆虫40种及发病的环境条件。     

Effects of arbuscular mycorrhizal fungi on the drought tolerance of Cyclobalanopsis glauca seedlings under greenhouse conditions

Cyclobalanopsis glauca is an important afforestation tree species that is widely used for revegetating the karst region of southwest China. Vegetation in this region is regularly commonly subjected to drought stress because of the geology and water shortages. Here, we investigated the influence of two arbuscular mycorrhizal fungi (AMF) Glomus mosseae and Glomus intraradices on the drought tolerance of C. glauca seedlings under greenhouse conditions. AMF-treated and non-AMF-treated C. glauca seedlings were maintained under two different water regimes (well watered: 80 % field capacity; drought stress: 40 % field capacity) for 90 days. The AMF colonization rate was higher under well-watered conditions compared to drought stress conditions. The growth and physiological performance of C. glauca seedlings were significantly affected by drought stress. Under drought stress conditions, mycorrhizal seedlings had greater height, base diameter, leaf area, and biomass compared to non-mycorrhizal seedlings. In addition, under drought conditions, AMF-inoculated seedlings had greater superoxide dismutase and peroxidase activity, higher soluble sugar content, and lower proline content compared to non-inoculated seedlings. Furthermore, AMF colonization increased the phosphorus and potassium content of seedling shoots under both well-watered and drought stress conditions. Therefore, AMF colonization enhanced the drought tolerance of C. glauca seedlings by improving growth performance, nutrient content, the quantity of osmotic adjustment compounds, and antioxidant enzyme activity. The results indicate that AMF are of potential use for the restoration of vegetation in the karst region of southwest China.     

Growth and survival of seedlings of various picea species under northern climatic conditions

The main purpose of these studies was to investigate the adaptation of young seedlings of various seed lots of Picea abies (L.) Karst., P. glauca (Moench) Voss, P. x lutzii Little and P. sitchensis (Bong.) Carr. to northern climatic conditions. Effects of temperature and photoperiod on elongation growth and growth cessation were studied under controlled conditions in a phytotron. In addition, growth and survival of the seedlings outdoors at 69°39’ N lat. were followed for two years. Seed lots of /’. abies originated from northern Norway (66° N lat.), those of the other species were from Alaska (between 57°.and 66° N lat.). The critical photoperiod for budset was 19–20 h for seed lots of P. abies. In general, the critical photoperiod for budset increased with increasing latitude of the seed source, but the results indicated a significantly shorter cirtical photoperiod for seed lots from about 60° N lat., <120 m a.s.l. of P. sitchensis (between 12 and 16 h) than for comparable seed lots of P x lutzii (17–18 h) or P. glauca (18 h). The time course of budset under natural light conditions, both in the phytotron and outdoors, generally followed the pattern predicted from the critical photoperiod. However, in P. glauca the budset occurred earlier than in P. abies although the latter had a longer critical photoperiod. Due to the short critical photoperiod and consequently delayed growth cessation and hardening, all seed lots of P. sitchensis (from 58° to 60° N lat.) were severely damaged during winter. Some damage was also observed in P. x lutzii and in P. abies. The optimum temperature for elongation growth was higher for P. sitchensis than for the other species. In the phytotron experiments, seedlings of P. sitchensis grew best at temperatures between 12 and 21°C, but at 9°C the best growth was obtained in some seed lots of P. abies. After two growth seasons outdoors, all seed lots of P. abies were taller than any seed lot of the other species. Also P. glauca seed lots and one seed lot of P. x lutzii showed good growth, and their growth rhythm seemed to be well adapted to the northern conditions.     

Avirulent isolate of the pinewood nematode Bursaphelenchus xylophilus,survives 7 months in asymptomatic host seedlings

An avirulent isolate (C14‐5) of the pinewood nematode, Bursaphelenchus xylophilus, was inoculated onto 45 seedlings of the susceptible host Japanese black pine (Pinus thunbergii) and its viability was investigated. The nematode survived inside host seedlings for approximately 7 months even when the host seemed to overcome the infection based on lack of foliar wilting and the observation of normal oleoresin flow.     

Insect vectors of the pinewood nematode: a review of the biology and ecology of Monochamus species

Pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970 , is a serious threat to susceptible pine forests of the world. The PWN is primarily vectored by Monochamus species (Coleoptera: Cerambycidae). The first occurrence of PWD was reported from Japan in the early 1900s. Following this report, Japanese scientists documented the community of bark‐ and phloem‐inhabiting insects associated with the nematodes in dying trees to determine possible vectors of the nematode. Monochamus alternatus was reported to be the most effective vector in Japan. The primary vector in North America is Monochamus carolinensis, and in Europe, it is Monochamus galloprovincialis. Further studies have been expanded through the nematode‐invaded countries of Korea, Taiwan, China and Portugal. There is an interspecific association between the PWN and its insect vectors, and it is an obligatory component of the disease cycle. It is crucial to understand this relationship as well as the population ecology of the beetle to aid in monitoring and control of this worldwide threat to pine forests. Studies to date indicate a remarkable similarity among beetle species around the globe for a variety of life‐history traits, including lifespan, adult emergence numbers, flight capability, nematode transmission rates and attraction to pine volatiles. Wherever pines are found, there is a beetle species capable of transmitting the nematode. Although flight performance and range is generally poor for this group of beetle vectors, the cryptic nature of the species and the lack of interest in the beetles by countries in the absence of the nematode have led to the disease establishing a foothold in a variety of countries such as Portugal. In this paper, studies conducted in different countries on Monochamus vector species of the PWN are compared and discussed.     

Surveys for asymptomatic persistence of Sphaeropsis sapinea on or in stems of red pine seedlings from seven Great Lakes region nurseries

The fungus Sphaeropsis sapinea persists on or in stems of asymptomatic red pine (Pinus resinosa) nursery seedlings, and proliferates to cause collar rot and mortality after planting. In the spring of 2002, seven nurseries were surveyed to determine the potential range in frequency of asymptomatic persistence: three operated by Wisconsin Department of Natural Resources (DNR), two by Minnesota DNR, one by Michigan DNR, and one by USDA Forest Service (in Michigan). At each nursery five groups of 20 asymptomatic red pine seedlings were collected near an inoculum source (red pine windbreak), if present, and five groups of 20 asymptomatic seedlings were collected away from such a source (1400 seedlings total). A segment of the lower stem/root collar from each seedling was surface disinfested and incubated on tannic acid agar. Transfers were made from resulting colonies and the pathogen identified from pycnidia and conidia produced in culture. The pathogen was identified from asymptomatic seedlings collected in all Wisconsin and Minnesota nurseries, but was never detected from seedlings from the Michigan DNR or USDA Forest Service nurseries. Frequencies of detection were greater (as high as 88%) from asymptomatic seedlings near red pine windbreaks including diseased trees than from seedlings distant from such windbreaks. A subset of isolates from asymptomatic seedlings was characterized using inter‐simple sequence repeat–polymerase chain reaction analysis. Most isolates were the A group of S. sapinea, but B group isolates (recently named Diplodia scrobiculata) were also obtained from one nursery. One Minnesota nursery was more extensively sampled in 2003, with 17–44 groups of five asymptomatic red pine seedlings collected in four separate fields (525 seedlings total). The mean frequency of detection of the pathogen in these four fields ranged from 40 to 71%. Persistence of S. sapinea on or in asymptomatic seedlings continues to be problematic, not only because of the potential for subsequent seedling mortality, but also as a means by which a pathogen may be widely distributed.     

Pine wilt disease: detection of the pinewood nematode (Bursaphelenchus xylophilus) as a tool for a pine breeding programme

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS‐PCR‐based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD.     

Impact of the invasion of pinewood nematode and the following different removal disturbance intensities on the plant diversity of Masson pine community

Reported in this paper are our findings of the study on changes in species diversity of different Masson pine communities after the invasion of Bursaphelenchus xylophilus (Steiner and Buhren) Nickle and subsequent and different removal disturbance management approaches. Based on the results, the species diversity indices for the arbor layers of the different communities can be arranged in the following order (from high to low): 1) broad-leaved stand after the removal of all infected pine trees in the pure pine stand in Fuyang; 2) lightly infected Masson pine and Schima superba mixed stand in Fuyang; 3) uninfected stand mixture of Masson pine and Castanopsis fargesii as a control; 4) lightly infested pure Masson pine stand in Fuyang; 5) Quercus variables stand formed following the selective removal of infected pine trees from a mixed Masson pine and Q. variables stand in Zhoushan Islands; 6) pure young Masson pine stand formed following the removal of all infected pine trees from a pure Masson pine stand; 7) pure Liquidambar formosana stand after the removal of infected pine trees from a pure pine stand in Zhoushan Islands; 8) a mixed stand consisting of Pinus thunbergii and the Masson pine in Zhoushan Island; and 9) moderately infected Masson pine stand in Zhoushan Islands. All the three diversity indices (R ₀ = richness, H′ = Shannon-Wiener index, and E = evenness) for the shrub layer did not show any significant differences among the various communities except for the pure pine stand in Zhoushan Island, which had the lowest diversity. The three indices for the herb layer of the pure young Masson pine, Q. variables stand, and L. formosana stand were higher than that of other stands. The integrated analysis showed that the Masson pine forest in different geographical situations and extent of damage had distinct disparity, just as in different disturbance degrees and restoring manners. We created the “index of disturbing intensity of stump and fallen woods” (IDISFW) to represent the degree of disturbance of tree removal to plant diversity. We found that the relationship between the species diversity index and the IDISFW shows both a lower and higher degree of removal disturbance that follows the “mid-altitude bulge” theory. Specifically, both excessive and insufficient removal of infected trees will cause a decline of plant species diversity to a certain degree. Covariance analysis indicated that different IDISFW had no significant effects on the species diversity of the arbor layer, but had different impacts on that of the shrub and herb layers, and therefore can be used to assess changes in species diversity of different Masson pine communities after the invasion of pine wood nematode. __________ Translated from Chinese Journal of Applied Ecology, 2006, 17(7): 1,157–1,163 [译自: 应用生态学报]     

Susceptibility of six pine species to Gremmeniella abietina isolates from Spain

Inoculation experiments were performed in order to evaluate the virulence of Gremmeniella abietina isolates from Spain on the main pine species planted in the Iberian Peninsula, as well as the influence of seedling age on this virulence. Two different experiments were carried out with four isolates of G. abietina from Spain. The greenhouse experiments consisted of seedling inoculations. One‐ and 2‐year‐old seedlings of the following five pine species were used: Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus sylvestris and Pinus uncinata; also, 1‐year‐old seedlings of P. nigra were inoculated. The relative necrosis length (RNL) caused by the pathogen after 130 days was used as a response variable. The laboratory experiments were performed on 2‐ to 6‐year‐old internodes of the above pine species excluding P. uncinata. The necrosis length after 6 weeks of incubation was measured. The results have shown that all G. abietina isolates were pathogens on seedlings of these six pine species and seedlings of P. halepensis were consistently the most susceptible ones, although it is important to take into account that all the isolates used in the present work were isolated from P. halepensis, the only pine species in Spain where G. abietina has been recovered up to now. The susceptibility of the other pine species depended on the age of the seedlings.     

Ecophysiology of seedlings of three Mediterranean pine species in contrasting light regimes

Seasonal dynamics of net photosynthesis (Anet) in 2-year-old seedlings of Pinus brutia Ten., Pinus pinea L. and Pinus pinaster Ait. were investigated. Seedlings were grown in the field in two light regimes: sun (ambient light) and shade (25% of photosynthetically active radiation (PAR)). Repeated measures analyses over a 12-month period showed that Anet varied significantly among species and from season to season. Maximum Anet in sun-acclimated seedlings was low in winter (yet remained positive) and peaked during summer. Maximum Anet was observed in June in P. pinea (12 micromol m-2 s-1), July in P. pinaster (23 micromol m-2 s-1) and August in P. brutia (20 micromol m-2 s-1). Photosynthetic light response curves saturated at a PAR of 200-300 micromol m-2 s-1 in winter and in shade-acclimated seedlings in summer. Net photosynthesis in sun-acclimated seedlings did not saturate at PAR up to 1900 micromol m-2 s-1 in P. brutia and P. pinaster. Minimum air temperature of the preceding night was apparently one of the main factors controlling Anet during the day. In shade-acclimated seedlings, photosynthetic rates were reduced by 50% in P. brutia and P. pinaster and by 20% in P. pinea compared with those in sun-acclimated seedlings. Stomatal conductance was generally lower in shaded seedlings than in seedlings grown in the sun, except on days with a high vapor pressure deficit. Total chlorophyll concentration per unit leaf area, specific leaf area (SLA) and height significantly increased in P. pinea in response to shade, but not in P. pinaster or P. brutia. In response to shade, P. brutia showed a significant increase in total chlorophyll concentration but not SLA. Photosynthetic and growth data indicate that P. pinaster and P. brutia are more light-demanding than P. pinea.     

Inoculation of pine stumps with Peniophora gigantea by chainsaw felling

By adding oidia of Peniophora gigantea to the lubricating oil of a chainsaw, stumps were automatically inoculated as the trees were felled. An experiment on Pinus sylvestris showed that direct chainsaw inoculation gave comparable results in terms of abundance of P. gigantea and control of Fames annosus, as conventional inoculation of stumps after felling.     

Quantifying biological nitrogen fixation of agroforestry shrub species using 15N dilution techniques under greenhouse conditions

Some land-use systems in Saskatchewan, Canada include the nitrogen-fixing trees buffaloberry (Shepherdia argentea Nutt.), caragana (Caragana arborescens Lam.) and sea buckthorn (Hippophae rhamnoides L.). These species provide various ecological functions such as ameliorating soil moisture, light and temperature but little work has been done quantifying biological nitrogen fixation by these species. Greenhouse experiments were conducted to quantify N₂-fixation using the ¹⁵N natural abundance and the ¹⁵N dilution methods. Buffaloberry failed to form nodules in all but one of the four replicates in the natural abundance experiment. Using the ¹⁵N dilution method, the percentage of N derived from atmosphere (%Ndfa) in the shoot of buffaloberry averaged 64 %. For caragana, the mean  %Ndfa was 59 and 65 % and seabuckthorn was 70 and 73 % measured using the natural abundance and dilution methods, respectively. Because of large variability in biomass production between plants grown in the natural abundance experiment and the dilution experiment, the amounts of N₂ fixed also were very variable. Buffaloberry fixed an average of 0.89 g N m^?2; the average for caragana ranged from 1.14 to 4.12 g N m^?2 and seabuckthorn ranged from 0.85 to 3.77 g N m^?2 in the natural abundance and dilution experiments, respectively. This corresponds to 16 kg N ha^?1 year^?1 for buffaloberry; an average of 15–73 kg N ha^?1 year^?1 in caragana and 11–67 kg N ha^?1 year^?1 in seabuckthorn. The substantial amounts of N₂ fixed by these species indicate that they have the potential to contribute to the overall N balance in land-use systems in which they are included.