Bursaphelenchus xylophilus causes pine wilt disease in susceptible pine species. To determine whether B. xylophilus persists in forests containing Abies sachalinensis and Picea jezoensis, we performed inoculation experiments using ten or 15 seedlings of A. sachalinensis, P. jezoensis, Pinus densiflora and P. thunbergii. Inoculation of 15 000 B. xylophilus caused 20 and 7% mortality in A. sachalinensis and P. jezoensis seedlings, respectively, in the nursery, indicating low susceptibility of A. sachalinensis and a lack of susceptibility of P. jezoensis. By contrast, B. xylophilus caused 40 and 70% mortality in P. densiflora and P. thunbergii, respectively. Bursaphelenchus xylophilus was recovered at extremely low densities from stems of externally asymptomatic seedlings of the first two tree species 9.5 months after inoculation. Inoculation of B. xylophilus on excised stem sections of A. sachalinensis and P. jezoensis seedlings showed a greater increase in population density in the bark than xylem after 3 weeks, whereas that on P. densiflora stem sections showed no difference in nematode density between the two tissues. The results suggest that B. xylophilus may persist in forests containing A. sachalinensis and P. jezoensis, when there are insect vectors with affinity for the nematode.
Two species-specific PCR primer pairs were developed for identifying the two
nematode species, Bursaphelenchus xylophilus and B. mucronatus. The primer
pairs were developed from the sequence of ribosomal DNA (rDNA) repeats to
produce DNA fragments of different lengths by PCR amplification. The DNA
fragments for B. mucronatus and B. xylophilus were 210 bp and 557 bp,
respectively. When mixed, neither primer pair inhibited the PCR
amplification of the other. Five isolates of B. xylophilus and four isolates
of B. mucronatus showed different band profiles of PCR products between the
two species, but identical profiles among isolates of the same species.
Differences among four isolates of Bursaphelenchus xylophilus and one of B.
mucronatus in vulnerability to the inhibitory effect of Pinus densiflora on
nematode dispersal were investigated by inoculating boiled and living branch
sections 5 cm long. The intrinsic dispersal ability and vulnerability to the
inhibitory effect of living branches differed among isolates. Additionally,
the degree of branch inhibition of nematode dispersal was examined by
inoculating the five isolates on 2.5 and 5.0 cm long, P. densiflora living
branch sections. An increase in section length induced an 80% decrease in
the number of nematodes of all isolates passing through the branch. The
extent to which passing nematode number decreased as branch section length
increased can be used to express susceptibility of pine species to pine wilt
disease. Virulence had no relation to intrinsic and realised dispersal rates
in B. xylophilus.
The effect of nematode infestation on dispersal and survival of both host plant and parasitic nematode was investigated experimentally using rice plants, Oryza sativa L. and Aphelenchoides besseyi Christie, a seed-borne ectoparasite. Nematode-inoculated plants produced a greater proportion of light seeds floating on water than non-inoculated plants. Nematode mortality was greater in light seeds than in heavy seeds. In the case of light seeds, the mean degree of seed swelling increased as the number of nematodes harboured increased, whereas it was constant with increasing numbers of nematodes for the intermediate and heavy seed groups. A smaller proportion of light seeds germinated and these took longer for seminal roots to protrude than from heavy seeds, irrespective of whether they derived from nematode-inoculated or non-inoculated plants. These results indicate a trade-off for rice seed specific gravity between dispersal and competition of rice seeds and another trade-off between dispersal and reproduction of nematodes harboured in the seed.
Bursaphelenchus mucronatus is closely related to Bursaphelenchus xylophilus,
the causative agent of pine wilt disease. Both nematodes are transmitted
between host pine trees as the fourth-stage dispersal juveniles (JIV) by
insect vectors. After the invasion of Japan by B. xylophilus, B. mucronatus,
native to Japan, appears to have been replaced in the pine forests during
the spread of the disease. To help understand this species replacement, the
number of JIV carried by an insect vector (the initial nematode load) was
compared between the two nematode species by using the beetle, Monochamus
alternatus, in the laboratory. The initial load of B. mucronatus was
significantly smaller than that of B. xylophilus although the number of
third-stage dispersal juveniles (JIII) concentrated at the pupal chambers
did not differ. Statistical analysis showed that the proportion of JIII
moulting to JIV was the most important among three components explaining the
difference in the initial load of B. mucronatus while the number of JIII
concentrated at the pupal chamber was the most important for B. xylophilus.
The phoretic affinity between the nematode and its vector is discussed in
relation to its role in the species replacement.
Monochamus alternatus adults carry the pine wood nematode Bursaphelenchus xylophilus. The nematodes are present in the tracheal system of vector beetles. It is known that carrying more than 10 000 nematodes causes drastic reduction in the lifespan and flight activity of beetles. To understand the effects of heavy nematode loads, 41 newly emerged M. alternatus adults were examined for lateral distribution of nematodes within the insect body. The nematodes were distributed unevenly between right and left sides of the beetle body at small nematode loads, whereas they were distributed evenly between them at heavy nematode loads. An analysis using Hagen-Poiseuille flow in a thin tube suggested that the rate of air flow decreased exponentially as the number of nematodes increased in a trachea. Thus, even distribution of nematodes between right and left sides of the vector body was considered to be responsible in part for the effects of heavy nematode loads on beetle activity.
Bursaphelenchus xylophilus is the pathogen associated with pine wilt disease
(PWD), an infectious disease of pine trees transmitted by cerambycid beetles
of the genus Monochamus. Bursaphelenchus xylophilus is an invasive species,
whilst B. mucronatus is a native congener and non-pathogenic to pine trees
in Japan. To provide experiment evidence of the biotic and/or abiotic
resistance to PWD expansion in a cool area of Japan, we inoculated B.
xylophilus into healthy pine trees in a Japanese red pine (Pinus densiflora)
stand with the indigenous pine tree-B. mucronatus-insect vector system
before invasion of B. xylophilus in 1993 and 1994. Extremely cool air
temperature with high precipitation and extremely high air temperature with
low precipitation were observed in the summers of the 2 years of
inoculation, respectively. The cool summer induced a low incidence of PWD
and delayed disease development, resulting in the replacement of B.
xylophilus by B. mucronatus within diseased trees and the emergence of
Monochamus saltuarius beetles carrying B. mucronatus from the trees 2 years
after the inoculation. The hot summer induced disease development in trees
in the year of inoculation, but such diseased trees did not become infection
sources because of the lack of M. alternatus, whose oviposition was
synchronised with the period of disease development in pine trees. This
study indicated that biotic factors were important in the inhibitory
mechanism in a pine forest against the spread of PWD in the stand. Relevant
biotic factors were the lack of M. alternatus and the presence of M.
saltuarius carrying B. mucronatus.
This study aimed to determine the spatial distribution patterns of Aphelenchoides besseyi among Oryza sativa seeds on panicle, plant hill, and paddy field spatial scales and to present a three-stage sampling method for estimating the mean density per seed in paddy fields. Living and dead nematodes were extracted individually from 20 seeds sampled from each of five panicles, which were sampled from each of six rice plant hills in each of eight paddy fields, where all plants had leaves exhibiting the 'white tip' symptom. Nested ANOVA indicated that A. besseyi density per seed was significantly different among the eight paddy fields, among rice plant hills in paddy fields, and among panicles in rice plant hills. The proportion of nematode-infested seeds (prevalence) increased and reached an upper limit as the mean density per seed on the panicle scale increased, whereas linear relationships were observed between nematode prevalence and the mean density on plant hill and paddy field scales. Relationships between mean density and mean crowding of nematodes per seed indicated that the nematodes exhibited clumped distribution on each of panicle, plant hill and field scales. Using these relationships, a three-stage sampling plan for estimating nematode density per seed at a specified precision level is presented.
To establish a simple, time-saving method for selecting Pinus tree candidates tolerant to the infection of Bursaphelenchus xylophilus, the causative agent of pine wilt disease, three experiments were conducted. When two virulent isolates Sc-9 and T-4 were inoculated on cut ends of branch sections, more nematodes were inhibited from passing through P. taeda branch sections than P. thunbergii sections. Sc-9 tended to pass through P. thunbergii sections more easily than T-4. The probability of nematodes passing through 5-cm-long P. thunbergii branch sections was greater when 200 nematodes were inoculated. When 200 Sc-9 nematodes were inoculated on 5-, 10- or 15-cm-long branch sections of P. taeda trees and P. thunbergii clones of different tolerance classes in late August, an insignificant correlation was found between the tolerance class of P. thunbergii clones and the number of nematodes passing through 5-cm-long branch sections, although nematode dispersal ability could distinguish between tolerant and susceptible pine clones in mid-August, suggesting it was a helpful method for selecting tolerant tree candidates. Abrupt seasonal increases in tolerance level suggested that weather conditions, such as hot temperatures and drought, may have disrupted the tolerance mechanism of resistant species of P. taeda.