Genetic variation in susceptibility to pine wilt disease of maritime pine (<Emphasis Type="Italic">Pinus pinaster</Emphasis> Aiton) half-sib families

Key message

This paper presents a greenhouse study for assessing the genetic variation in maritime pine (Pinus pinaster Aiton) in response to pinewood nematode (Bursaphelenchus xylophilus (Steiner et Buhren) Nickle), which is a causal agent of pine wilt disease. Fifteen out of 96 half-sib families were selected as less susceptible. This experiment is an important first step for creating a resistance breeding program.

Context

Pine wilt disease is caused by the pinewood nematode (Bursaphelenchus xylophilus (Steiner et Buhren) Nickle), a quarantine pest, and is a concern to maritime pine (Pinus pinaster) in Portugal due to its economic, environmental, and social impacts. This disease is regarded as a major threat to European forests.

Aims

This paper aimed to evaluate the genetic variation in maritime pine families that were inoculated with pinewood nematode, identify the most resistant families, and establish the guidelines for a resistance improvement program.

Methods

Two-year-old half-sib progenies obtained from 96 plus trees were inoculated. The plants were monitored for survival on four different dates. The statistical analysis followed the mixed model theory.

Results

Genetic variability of the susceptibility to pine wilt disease was observed. At 157 days after inoculation, the 15 highest genetic ranking families out of 96 were selected, having a predicted survival mean of 15.6% instead of 11.0% on average for the all 96 families.

Conclusion

This study allows for the implementation of an improvement program to help control pine wilt disease.

     

Susceptibility of adult trees of the endangered species Pinus armandii var. amamiana to pine wilt disease in the field

Adult trees of Pinus armandii var. amamiana (PAAm) and P. thunbergii grown in the field were inoculated with 100000 or 1000 of the nematode Bursaphelenchus xylophilus to evaluate their susceptibility to pine wilt disease. PAAm trees inoculated with 100000 nematodes started to show disease symptoms 2 weeks after inoculation, and all died within 29 weeks. Although the PAAm trees inoculated with 1000 nematodes tended to show delayed disease symptoms compared with those inoculated with 100000 nematodes, all of them died within 33 weeks after inoculation. All P. thunbergii trees inoculated with 1000 nematodes had died 6 weeks after inoculation. In the nematode-inoculated PAAm trees, death of branches distal to the nematode inoculation site was the first visible symptom, followed by the systemic discoloration of needles, whereas the whole tree wilted simultaneously in P. thunbergii trees. In nematode-inoculated PAAm trees, the period from inoculation to death was longer than that in P. thunbergii. These results suggest that adult PAAm trees are susceptible to pine wilt disease, but are less vulnerable than P. thunbergii.     

Statistical interferometric investigation of nano-scale root growth: effects of short-term ozone exposure on ectomycorrhizal pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) seedlings

This study presents the effects of short-term ozone exposure on the nano-scale growth behavior of the fine roots of Pinus densiflora (Japanese red pine) seedlings. Root elongation measurements were obtained in nanometers for very short (sub-second) time intervals by using the optical interference method called statistical interferometry, developed by the authors. Three categories of P. densiflora seedlings were investigated; two categories were infected with ectomycorrhiza of Pisolithus sp. (Ps) and Cenococcum geophilum (Cg), while the third was without any fungal infection. In experiments, two points on a root with a separation of 3 mm were illuminated by laser beams and the elongation was measured continuously by analyzing speckle patterns successively taken by a CCD camera. The ectomycorrhizal fungi-infected and uninfected seedlings were exposed to ozone at concentrations of 120 and 240 ppb for periods of 1, 3, or 5 h in separate treatments. The root elongations of P. densiflora seedlings were measured before and immediately after the each ozone treatment and then the root elongation rates (RER) were determined for growth-measurement periods of 5.5 s and 9.5 min. From the measurements obtained for 9.5 min, we found that the RERs of uninfected and Cg-infected seedlings were reduced by 42 and 18%, respectively, after 5 h of exposure to 120 ppb ozone compared with that before exposure, while the reduction in RER of Ps-infected seedlings was not significant. When the concentration of ozone was increased to 240 ppb, the RERs of Ps-infected and Cg-infected seedlings were reduced by 32 and 44%, respectively, after exposure for 5 h, while the reduction in RER of uninfected seedlings was 59%. These observations prove that the non-mycorrhizal seedling roots are more sensitive to ozone stress. From this study, we found that the RERs of both mycorrhizal and non-mycorrhizal seedlings apparently fluctuated throughout the measurements, even within a few minutes.     

Survival ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture severely damaged byBursaphelenchus xylophilus

The total area ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture in 1978 was 65,200 ha, which decreased to 30,300 ha by 1985 mainly due to pine wilt mortality caused byBursaphelenchus xylophilus. This damage has also continued thereafter. To estimate the survivability of pine trees in Ibaraki Prefecture, pine tree mortality has been studied in eight experimental forests for over 20 years, and ground surveys throughout the Prefecture were also conducted in 1995. Survival in the experimental forests corresponded well to the results of ground surveys. Pine forests remained as pure stands if control measures were undertaken or if they were located in cool areas. In warm areas where no control was undertaken, most of the pine forests disappeared and only a few pine trees remained in mixed forests, while on dry soils no mature or old pine trees survived. Since surviving pine forests are often cut for wood utilization,P. densiflora andP. thunbergii may decrease in area to become rare species in the future unless controls are applied and/or reforestation with resistant pines is carried out. A part of this paper was orally presented at the 108th Annual Meeting of the Japanese Forestry Society (1997).     

Differential responses of Central American and Mexican pine species and Pinus radiata to infection by the pitch canker fungus

Seedlings from a wide array of 23 species, varieties,and geographic races were screened for resistance topitch canker using artificial inoculation in agreenhouse. Seed to represent these taxa weregenerally collected in natural stands. In addition,seedlings from 79 families of P. radiata fromcommercial populations from Chile and New Zealand werescreened in a separate experiment. There was littlevariation in resistance among the commercial P.radiata families, with over 98% mortality. Similarresults were obtained with native P. radiatapopulations. All close relatives of P. radiata(sub-section Patula) were very susceptible. However,a number of closed-cone pine species (specifically,from sub-section Oocarpa) were generally veryresistant, with survival approaching 100%. Inaddition, in P. oocarpa, P. jaliscana, and P. tecunumanii from low elevation provenances anextremely high frequency of trees suffered no apparentdamage from the pathogen (92%, 85% and 80%,respectively).     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Variation in susceptibility of six pine species and hybrids to pitch canker caused by Fusarium circinatum

Six pine species or hybrids were tested for susceptibility to pitch canker caused by Fusarium  circinatum. Pinus  densiflora, Pinus  thunbergii, Pinus  x rigitaeda (Pinus  rigida × Pinus  taeda), P. rigida × P. x rigitaeda, Pinus  echinata and Pinus  virginiana were inoculated with three spore loads (50, 500 and 5000 per tree) of F. circinatum. External symptoms, lesion length, and the frequency of reisolation of the fungus were investigated. External symptoms were greatest in P. echinata, followed by P. virginiana, however, P. densiflora was not susceptible to F. circinatum. Based on mean lesion lengths, the six pine species or hybrids differed significantly (p < 0.01) in susceptibility to pitch canker. Pinus  echinata sustained the longest lesions, whereas P. densiflora sustained the shortest lesions. The effect of inoculum density was not significant among three spore treatments within species (p = 0.17), although lesion length was slightly greater at higher spore loads over all pine species. The fungus was reisolated from inoculated stems of all pine species tested, even on trees showing little or no damage from the disease. Additional studies are needed to further explore the basis for resistance to pitch canker.     

Pathogenicity studies with Bursaphelenchus mucronatus in Scots pine in Finland

The objective of the study was to determine the role of Finnish Bursaphelenchus mucronatus as a possible pathogen in Scots pine, Pinus sylvestris, under Finnish ambient weather conditions. Small seedlings and young field grown trees were used as inoculation targets. The results suggested the nematode being of minor importance as a tree pathogen in Finland.     

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.     

Role of asymptomatic carrier trees in epidemic spread of pine wilt disease

To determine why pine wilt disease caused by the pinewood nematode (Bursaphelenchus xylophilus) recurs in the same pine stand even after thorough eradication of dead pine trees, the amount of oleoresin exudation from artificial wounds was measured from 72 Pinus koraiensis trees, highly susceptible to this disease, for 4 years. The amount of exuded oleoresin was rated from 0 to 4. All values obtained for each tree were summed at each measurement; thereby a cumulative curve was drawn to monitor the physiological condition of each test tree. Cumulative curves suggest that some pine trees that died had already been infected in the previous year or earlier and then had survived without any visible symptoms. If cessation of oleoresin exudation delays, and overlaps with activity of Monochamus alternatus, the vector beetle of pinewood nematodes, in the following season, such trees can be referred to as latent carriers or asymptomatic carriers. They could play a significant role as attractants for M. alternatus that could then transmit B. xylophilus to neighboring trees. Behavior of M. alternatus caged with several pine seedlings, only one of which acted as an asymptomatic carrier of B. xylophilus, confirmed this idea. The presence of asymptomatic pine trees, which harbored B. xylophilus nematodes or had reduced annual elongation, near stumps of newly dead trees in the study stand also substantiated this hypothesis.     

Recessive inheritance of morphological characteristics of utsukushimatsu,<Emphasis Type="Italic">Pinus densiflora</Emphasis> f.<Emphasis Type="Italic">umbraculifera</Emphasis>

Pinus densiflora f.umbraculifera, commonly known as utsukushimatsu, is a distinctively shaped form of Japanese red pine whose growth is restricted to a forest stand in Shiga Prefecture, Japan. The inheritance mode of morphological characteristics of utsukushimatsu was studied to preserve the genetic resource of this pine. As previously reported, F₁ trees grown from open-pollinated seeds harvested from trees inhabiting the native stand showed two phenotypes: one resembling utsukushimatsu, which produces multiple trunks, and the other resembling normalP. densiflora, which produces one or a few trunks. In the present study, controlled pollination was carried out using F₁ and normalP. densiflora trees. Segregation ratios of the two phenotypes observed in the F₂ population showed that the morphological characteristics of utsukushimatsu are inherited recessively. This suggests that the mutation of one gene or a few closely linked genes controls the morphological characteristics of utsukushimatsu. Since multiple trunk formation of utsukushimatsu might be related to a loss of lateral bud inhibition, it follows that a simple gene mutation breaks apical dominance inP. densiflora.     

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.     

The effects of long‐term exposure to simulated acid rain on the development of pine wilt disease caused by Bursaphelenchus xylophilus

To elucidate the synergetic effects of acid rain on the development of pine wilt disease, we measured the sap flow rate in the stems and the chlorophyll content in the needles of 10‐year‐old Japanese black pine trees, Pinus thunbergii and 12‐year‐old Japanese red pine trees, Pinus densiflora, after exposure to simulated acid rain (SAR, pH 3) or tap water (TW, pH 6.3) as a control. The heat pulse method was used for the estimation of the sap flow rate. No apparent difference was found in the sap flow rate between the trees exposed to SAR and TW, but the chlorophyll content of needles at the end of the treatment was significantly higher in the trees exposed to SAR than in those exposed to TW. When the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease, was inoculated onto the Japanese black pines that had been exposed to SAR repeatedly for 1 year, the period to death was shortened. Japanese red pines that had been exposed to SAR for 2 years, however, did not show any development of symptoms after the nematode inoculation, suggesting that acid rain only affects pine wilt disease slightly, if at all.     

Physiological process of the symptom development and resistance mechanism in pine wilt disease

Pathophysiological changes during the symptom development of pine wilt disease are reconsidered from recent investigations. The symptom development is divided into two stages: the early and the advanced stages. In the early stage, small number of nematodes migrate in cortex, then in xylem of the stem, and induce denaturation and necrosis of parenchyma cells. These changes in parenchyma are regarded as defense reactions of pines which result in terpene synthesis in xylem cells and embolism in tracheids. Such changes in the early stage can be induced in both susceptible and resistant pine species by either virulent or avirulent isolates of pinewood nematode (Bursaphelenchus xylophilus), or byB. mucronatus. No change occur in physiological status of leaves, and nematode reproduction is suppressed during this stage. Pine trees can survive if symptom does not progress from this stage. The symptoms of the advanced stage usually occur only in susceptible pines infected by virulent nematode isolates. At the beginning of the advanced stage, enhanced ethylene production by stem which coincides with cambial destruction occurs, and results in embolism of the outermost xylem in the portion. The embolism causes decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, symptoms develop drastically with a burst of nematode population. There seems to be some unknown mechanism which suppress nematode reproduction and invasion to the cambial zone. This mechanism is thought to be photosynthesis-dependent, so that in photosynthesis-decrased conditions, even avirulent nematodes can multiply and invade cambium to induce tree death. Water stress in hot and dry summer should accelerates symptom development from the early to the advanced stage through such decrease of photosynthesis-dependent “cambial resistance”.     

Histopathology of primary needles and mortality associated with white pine blister rust in resistant and susceptible Pinus strobus

White pine blister rust caused by Cronartium ribicola is a damaging non‐native disease of five‐needled pines in North America. Efforts to control the disease and mitigate damage to date have been only somewhat effective. Recent efforts to improve the health of eastern white pine and reestablish the tree as a dominant species in the North Central United States have focused on identification and propagation of disease‐free eastern white pine (Pinus strobus) growing in areas with a high incidence of blister rust. Many of these selections have been shown to resist infection following artificial inoculation with C. ribicola. In this study, 13 eastern white pine families derived from controlled pollination of selections previously determined to possess putative resistance as well as susceptible selections were inoculated with C. ribicola. Mortality data from inoculation studies show superior survivability in three families with over 60% of seedlings able to survive the 52 week post‐inoculation monitoring period compared to 0–10% survival of the most susceptible families. Primary needles were collected for histological analysis from all inoculated families 4 weeks after inoculation and from selected families 6.5 weeks and 38 weeks after inoculation. Histological observations of infection sites show distinct resistance reactions in the families more likely to survive infection based on mortality data. Analysis of the reactions in susceptible families revealed extensive hyphal colonization of the vascular bundle and adjacent mesophyll cells that appear uninhibited by tree responses. In resistant families, collapsed cells adjacent to infection sites, heavy deposition of phenolic compounds and abnormal cell growth were documented more frequently and appear to play an integral role in the ability of these eastern white pine families to impede growth of C. ribicola in primary needle tissue.     

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.     

Comparison of the gene expression profiles of resistant and non‐resistant Japanese black pine inoculated with pine wood nematode using a modified LongSAGE technique

The Japanese black pine (Pinus thunbergii) is highly susceptible to pine wilt disease caused by the pine wood nematode (PWN; Bursaphelenchus xylophilus). To cope with this disease, researchers and tree breeders selected PWN‐resistant individuals in a previous breeding program. In an attempt to understand the mechanisms of resistance in the Japanese black pine, we created four LongSAGE (serial analysis of gene expression) libraries. A total of 20 818 tags were studied, including 5194 tags from a PWN inoculated resistant pine, 5218 a non‐inoculated resistant pine, 5194 an inoculated non‐resistant pine, and 5212 a non‐inoculated non‐resistant pine. The analysis of the libraries indicated that 14 tag species were significantly up‐regulated (e.g., pathogenesis‐related proteins 2 and 4, osmotin, lipoxygenase, and chalcone synthase), and nine were down‐regulated (eukaryotic translation initiation factor SUI1, translationally controlled tumor protein, and xyloglucan endotransglycosylase) by the PWN inoculation in both the resistant and non‐resistant pines. On the other hand, 38 tag species were significantly expressed at a higher level only in the resistant pine (catalase, dienelactone hydrolase family protein) and 25 were expressed at a higher level in the non‐resistant pine (pathogenesis‐related proteins 1, 2, and 3, and leucoanthocyanidin dioxygenase). These differentially expressed genes are presumed to reflect some of the differences between the resistant and non‐resistant pines. Our results provide valuable information on the complex responses induced in the resistant and non‐resistant pine trees in response to PWN invasion.     

The role of litter and humus layer as a mortality factor of seedlings of Japanese red pine,Pinus densiflora Sieb. et Zucc.

The process of decrease in seedlings of Japanese red pine (Pinus densiflora Sieb.et Zucc.) and the effect of litter and humus layers on the mortality were studied in their stand and adjacent bare land. After the rainy season, only small number of individuals died in the bare land but more seedlings died in the forest. The number of seedlings killed by drought was greater in the forest than in the bare land. The population of naturally occurring current year seedlings was larger in the forest than in the bare land, but that of 1- to 5-year-old seedlings was smaller. Severe summer drought exceeding the permanent wilting point was observed only in the forest F-H horizon where greater part of seedling roots distributed, this may be one of major mortality factors. Inhibitory substances to seedling growth in fresh fallen pine needles were also suggested.     

Impact of invasion of pine wood nematode on the growth of dominant shrub Pleioblastus amarus in Pinus massoniana communities

When pine trees are invaded by pine wilt diseases, the severely infected pine trees will die and fall down, or they will be removed when found to be damaged by the disease. It gives rise to the invasion of other species in these empty niches originally occupied by pine trees, i.e., competing surrounding trees or understory shrubs will invade the empty niches during the following years. As a result, the spatial distribution and pattern of the main tree species in a pine forest will change, and a niche variety in the main population will occur. In the end, the direction of the succession and restoration of the pine forest ecosystem will be affected. In our study, a Pinus massoniana forest with the dominant shrub, Pleioblastus amarus, was invaded by pine wood nematode and was clear cut. Selecting this community as our research object, we studied the effect of the invasion of the pine wood nematode on the growth of the dominant shrub, P. amarus, in this Pinus massoniana forest. Our results show that, after the attacked pine trees were removed, the niche was occupied by Pleioblastus amarus and other shrubs, which benefited the growth of P. amarus to its climax. Growth of P. amarus at the climax stage was greater compared with the unhealthy pine forest and the control group.     

Inoculation of seven pine species or hybrid seedlings with Korean isolates of pinewood nematode under greenhouse conditions

? Seedlings of seven pine species or hybrids, Pinus densiflora, Pinus thunbergii, Pinus virginiana, Pinus echinata, Pinus koraiensis, Pinus thunbergii × Pinus massoniana and Pinus × rigitaeda, were inoculated in a greenhouse with 100, 1 000 and 10 000 pathogenic nematodes, Bursaphelenchus xylophilus, to compare initial symptom development and to evaluate susceptibility to pinewood nematodes.

? Genetic types were significantly different in the starting day of needle discoloration (χ² = 43.48; P < 0.001) and the period from nematode inoculation to seedling death (χ² = 32.85; P > 0.001), but treatment groups were not statistically different in the days from inoculation to seedling death in any of the pine species.

? There was no relationship between seedling mortality and the number of nematodes recovered from infected seedlings. Only eight of the nematode-inoculated seedlings remained alive by day 83 after inoculation, including four seedlings each of P. echinata and P. × rigitaeda grown from bulked seed collections.

? Seedlings from the bulk lots were likely more genetically diverse than those from the single-source lots. A broader genetic representation of pine species or hybrids is suggested for screening for planting stocks resistant to B. xylophilus.