Inoculation of several Bursaphelenchus xylophilus group nematodes into adult trees of Pinus thunbergii and their survival in the trees

To clarify the pathogenicity of Bursaphelenchus nematodes to adult pine trees, inoculation experiments using six species of B. xylophilus group nematodes and ca. 10‐year‐old trees of Pinus thunbergii were conducted. Trees inoculated with an avirulent isolate (C14‐5) of B. xylophilus did not die during the survey, but showed a decline in oleoresin exudation compared with the controls. Fifteen months after the inoculation, a small number of B. xylophilus survived in a tree inoculated with B. xylophilus C14‐5. Trees inoculated with B. mucronatus, B. doui, B. luxuriosae, B. conicaudatus and Bursaphelenchus sp. NK224 (undescribed) showed no decline in oleoresin exudation and no external symptoms of wilt. However, 9 months after the inoculation, a small number of B. luxuriosae survived in a tree inoculated with the nematodes, although four other nematode species were not isolated from trees inoculated with them. These results were approximately consistent with our previous results (Kanzaki, N.; Aikawa, T.; Maehara, N.; Ichihara, Y., 2010, J. For. Res.; in press), in which an avirulent isolate (OKD‐1) of B. xylophilus and B. luxuriosae caused water flow inhibition without external symptoms in 3‐year‐old seedlings. Therefore, to examine the pathogenicity of the nematodes to pines, it is useful to use 3‐year‐old seedlings in inoculation experiments when adult trees cannot be used.     

Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree

To assess the role of bacteria in pine wilt disease (PWD), aseptic M form (with a mucronated tail) and R form (with a round tail) of Bursaphelenchus xylophilus and B. mucronatus were obtained and compared, in terms of reproduction and pathogenicity, with non‐aseptic nematode. In addition, bacteria isolated from non‐aseptic nematodes and pine trees inoculated with non‐aseptic nematodes were identified. The results indicated that the bacteria associated with nematodes significantly lowered the reproduction of R form of B. xylophilus and B. mucronatus. Both the non‐aseptic and aseptic R forms of B. xylophilus induced death in all infected 7‐ to 8‐year‐old pine trees, while the non‐aseptic and aseptic M forms of B. xylophilus and B. mucronatus caused almost no plant mortality. High numbers of the non‐aseptic and aseptic R forms of B. xylophilus were distributed throughout the inoculated trees, while B. mucronatus and M form of B. xylophilus nematodes were lower in number and their distribution in stems limited within the inoculation site. Bacteria isolated from non‐aseptic nematodes were not recovered from the pine trees inoculated with these same kinds of nematodes. Two species of bacteria were both isolated from non‐aseptic B. mucronatus and from R form of B. xylophilus. Microbacterium trichotecenolyticum was common to both the control and inoculated pine trees. These results suggest that R form of B. xylophilus is the causal agent of PWD and that bacteria cannot increase the virulence of B. xylophilus and B. mucronatus.     

Pathogenicity of Ceratocystis resinifera to Norway spruce

The blue‐stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single‐point inoculations on young trees (two inoculations per tree on 14‐year‐old trees) and mass‐inoculations on older trees (～200 inoculations per tree on 34‐year‐old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue‐stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue‐stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non‐pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.     

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.     

Rust resistance in Salix induced by inoculations with avirulent and virulent isolates of Melampsora larici‐epitea

Six Salix clones and four Melampsora larici‐epitea isolates were used in two leaf‐disc inoculation experiments to determine whether disease severity was affected by the presence of both virulent and avirulent rust genotypes. In the first experiment, an equal amount of urediniospores of a virulent isolate and an avirulent isolate was applied simultaneously using four levels of spore suspension. In the second, the willows were inoculated with one isolate first and then, after 3 days, with another using two spore concentrations. In the first experiment, overall rust spore production was reduced by 48.6% in inoculations with mixed inocula compared with that in the inoculations with single virulent isolates. In the second experiment, 20 of the 36 combinations involving pre‐inoculations with avirulent isolates showed significant reduction in spore production. The suppressive effects on rust sporulation were more obvious at the higher inoculum densities and on the clones S. × calodendron and S. × mollissima‘Q83’.     

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.     

A simple,polymerase chain reaction‐restriction fragment length polymorphism‐aided diagnosis method for pine wilt disease

For diagnosis of pine wilt disease, a simple PCR‐RFLP method was developed to identify and to differentiate two similar nematode species, based on a living or preserved single specimen. Pinewood nematodes, Bursaphelenchus xylophilus, and Bursaphelenchus mucronatus were examined. A single nematode in 1 µl of distilled water was put on a glass slide. When the water had almost dried the nematode was crushed with a filter paper chip, 1.5 mm × 1.5 mm, with the aid of forceps. The filter paper chip containing nematode remains was immediately placed into PCR buffer as the DNA template. The primer set used was to amplify ribosomal DNA containing the inter‐transcribed spacer (ITS) 1, 5.8S and ITS2 regions. The PCR product was consistently obtained from a single nematode, and digesting the product with restriction endonuclease, Hinf I, enabled discrimination between B. xylophilus and B. mucronatus. This method was simple, convenient and definitive, and could successfully determine the pathogen in the diagnosis of pine wilt disease. This method was applicable also to nematode specimens preserved under various conditions except in the case of those preserved in aldehyde‐containing fixatives.     

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

We investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.     

Ethylene production during symptom development of pine-wilt disease

Ethylene release from Pinus thunbergii Parl. seedlings was examined after inoculation with virulent and avirulent isolates of Bursaphelenchus xylophilus. Enhanced ethylene release was observed only in seedlings inoculated with the virulent isolate. The avirulent isolate induced necrosis of xylem ray cells and embolism in some tracheids, however, the cambium and outermost xylem was intact where water conduction was maintained. The ethylene increase coincided with cambial death in seedlings inoculated with the virulent isolate.     

Virulence of Bursaphelenchus mucronatus to pine seedlings and trees under field cownditions

Bursaphelenchus mucronatus is a parasitic nematode of pine that is widely distributed in the natural pine forests of Asia and Europe. It has a very similar morphology and biology to that of Bursaphelenchus xylophilus, the causal agent of pine wilt disease, but has generally been considered to be non‐pathogenic to pine. However, in some provinces of China, B. mucronatus has been isolated from dead pine trees rather than B. xylophilus. Previous studies have shown that B. mucronatus can induce the death of pine seedlings under glasshouse conditions. To investigate the virulence of B. mucronatus, 2‐year‐old seedlings of Pinus massoniana and Pinus elliottii were inoculated with one of six isolates of B. mucronatus under field conditions in April 2014 and their condition was monitored over a year. The virulence of the six B. mucronatus isolates differed on the three host species: P. elliottii seedling mortality ranged from zero to six of the 18 inoculated seedlings, whereas P. massoniana seedling mortality ranged from four to 12 of the 18 inoculated seedlings. Three B. mucronatus isolates that appeared to cause different levels of mortality among the seedlings were used to inoculate 12‐year‐old Pinus thunbergii trees in August 2014. The trees were monitored for a year, during which time between 4 and 12 of the 18 inoculated trees in each treatment wilted and died. The average monthly temperature during the test period appeared to be similar to that of the historical average in the test areas; however, both study sites experienced above‐average rainfall. This study demonstrated that B. mucronatus has potential virulence on pine trees and provided experimental evidence that high temperatures or drought stress is not essential for the virulence of B. mucronatus.     

Development of two reverse transcription‐PCR methods to detect living pinewood nematode,Bursaphelenchus xylophilus,in wood

Pinewood nematode, Bursaphelenchus xylophilus, is an inhabitant of native pine species of North America, where its presence in trees is non‐pathogenic. By contrast, the introduction of this nematode to forests overseas has devastated some pine stands and is recognized as a pest of phytosanitary concern by some countries' National Plant Protection Organizations. The ability to detect B. xylophilus in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of B. xylophilus rely on the presence of genomic DNA and thus will detect both living and dead nematodes without differentiation. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between live and dead B. xylophilus are critical. We have developed an endpoint RT‐PCR assay and a SYBR Green 1 real‐time RT‐PCR assay, both of which selectively identify living pinewood nematode by detecting the presence of Hsp70 mRNA as a viability marker. Both of these assays may help overcome or resolve disputes involving the detection of pinewood nematode at the port of entry and can also be used to evaluate the efficiency of wood treatment procedures.     

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”.     

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.     

Canadian conifers as hosts of the pinewood nematode (Bursaphelenchus xylophilus): Results of seedling inoculations

Seedlings of 22 species of conifers from across Canada were inoculated with m and r form isolates of the pinewood nematode (Bursaphelenchus xylophilus). In an experiment made under ambient (summer‐fall) temperatures in a shadehouse at Victoria, British Columbia, Canada, 8 of the 22 conifer species were killed by the nematodes, but mortality was low, i.e. 4–30%. Pines (eastern white, Jack and red) were more susceptible than other conifers. Yellow cypress, eastern white cedar, western red cedar and western hemlock were not killed by the nematodes. In a second experiment made at elevated temperatures (30°C‐16h long days, 25°C‐8 h long nights) in a greenhouse, 18 of the 22 conifer species died following nematode inoculation. Again, pines (lodgepole, eastern white, western white and red) were among the most susceptible tree species and the four conifers that were unaffected in the first experiment were not killed. Tamarack and western larch, both immune at ambient temperatures, were the two most susceptible conifers at elevated temperatures. Compared to ambient temperatures, seedlings at elevated temperatures died quicker and contained more nematodes. M and r form nematodes were equally pathogenic in both experiments.     

An inoculation experiment of Japanese <Emphasis Type="Italic">Bursaphelenchus</Emphasis> nematodes on Japanese black and red pine, <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">P. densiflora</Emphasis>

The pinewood nematode (PWN) Bursaphelenchus xylophilus is an invasive pathogen that was introduced from North America to Asian countries and Portugal and is devastating native pine forests. Some native European and Asian Bursaphelenchus nematodes also have weak to moderate pathogenicity to native pine species. To evaluate the potential risk of native Bursaphelenchus species, we inoculated ten Japanese Bursaphelenchus species into native pine species (the dominant forest species) in Japan, and evaluated their pathogenicity using mortality and tracheal tissue damage as indices. Inoculation was conducted on August 3, 2007, and the symptoms were observed every 2 weeks until February 1, 2008. None of the inoculated trees, excluding the pathogenic PWN inoculated control, showed external disease symptoms; however, four species [a less pathogenic PWN isolate, B. luxuriosae, Bursaphelenchus sp. NK215 (undescribed), and NK224 (undescribed)] caused tracheal tissue damage in inoculated seedlings and showed weak pathogenicity. Therefore, we conclude that there are some potentially pathogenic native species of nematodes distributed in Japan. Interestingly, two of these weakly pathogenic species, B. luxuriosae and NK215, are not associated with Pinaceae trees, suggesting that nematode pathogenicity may be a pre-adaptive character. More experimental studies under different conditions are necessary to accurately evaluate the potential risk of these pathogens.     

Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.     

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.     

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.     

Seasonal effect on infection and development of lesions caused by Cryphonectria parasitica in Castanea sativa

Seasonal variation in the development of chestnut blight, caused by Cryphonectria parasitica, was investigated by inoculating in situ chestnut trees and in vitro excised chestnut segments, at either monthly or 3‐monthly intervals throughout 30 months. Inoculations were made with conidia and mycelium of a virulent isolate and with mycelium of a hypovirulent isolate. Conidial inoculations of living sprouts or excised segments between May and July resulted in the greatest incidence of infection whereas inoculations in autumn and winter, in vitro as well as in situ, did not reveal any visible disease. However, from these symptomless inoculated stems, C. parasitica was isolated 3 months after inoculation. Inoculations with the mycelium of the virulent isolate always resulted in lesions, except in January 1999, and the greatest rate of lesion development occurred for inoculations made in the spring and summer. There was a significant seasonal effect on lesion development. Lesions caused by the hypovirulent isolate, smaller than those caused by the virulent isolate, followed a similar seasonal pattern. The same seasonal variations were observed for inoculations in vitro of excised segments. Relative water content (RWC) of chestnut bark significantly varied with bark sampling date. The rate of lesion development in sprouts significantly correlated with average minimum (ATn) and maximum (ATx) temperatures and the sum of rainfall during inoculation period, with the rate of lesion development measured in excised segments 10 days after inoculation (R10d) and with RWC measured on the day of inoculation. In multiple regression models, variables ATx and R10d best explained variation in lesion development.