Suppressive effects of Bursaphelenchus mucronatus on pine wilt disease development and mortality of B. xylophilus-inoculated pine seedlings

Nematology ◽  
2014 ◽  
Vol 16 (2) ◽  
pp. 219-227 ◽  
Author(s):  
Si-Mi Liao ◽  
Satomi Kasuga ◽  
Katsumi Togashi

Bursaphelenchus xylophilus causes pine wilt disease in Pinus trees whereas B. mucronatus has no or little virulence to the trees. Interspecific crossing experiments conducted so far suggest reproductive interference between the two nematode species. Theory predicts that one of the two competing species populations quickly displaces the other through reproductive interference in a frequency-dependent manner. Thus, it is anticipated that B. mucronatus suppresses the virulence of B. xylophilus against pine trees when B. mucronatus heavily outnumber B. xylophilus. To determine the suppressive effects of B. mucronatus, the two nematode species were inoculated simultaneously on 30 3-year-old Pinus thunbergii seedlings at three combinations of different numbers, and B. xylophilus alone was inoculated on 30 other seedlings at the corresponding numbers in early August. Seedlings were observed at intervals of 4 or 6 weeks and two stem sections were sampled from each seedling to determine the density and species composition of nematode populations after death or in December. Inoculation of B. mucronatus significantly retarded the speed of foliage discolouration from 0.170 ± 0.024 week−1 to 0.061 ± 0.017 week−1 and significantly prolonged the survival time of seedlings. Bursaphelenchus mucronatus significantly reduced the nematode density from 1799.7 ± 305.0 to 521.0 ± 148.4 (g dried seedling stem)−1. Analysis of rDNA genotype showed 1846 B xylophilus, no B. mucronatus and one hybrid.

Nematology ◽  
2006 ◽  
Vol 8 (6) ◽  
pp. 869-879 ◽  
Author(s):  
Kazuyoshi Futai ◽  
Natsumi Kanzaki ◽  
Yuko Takeuchi

AbstractPine wilt disease causes ecological and economic damage in Japanese pine forests in spite of intensive effort to protect them from the pine wood nematode, Bursaphelenchus xylophilus. Pine trees infected with B. xylophilus emit a characteristic bouquet of volatile compounds bioactive to the vector beetle of the nematode, Monochamus alternatus, and potentially affecting symptom development inside the trees. To investigate the qualitative and quantitative properties of volatile compounds in the field, we profiled the volatile emissions in two Japanese black pine stands, one naturally suffering from pine wilt disease and the other artificially inoculated with B. xylophilus. In both pine stands, the emission of some terpenoids from the infected trees such as (−)-α-pinene, began to increase in summer, overlapping the oviposition season of the vector beetle, but peaked in the summer and autumn. These data suggest that the beetles may not necessarily depend on the tremendous quantity of volatiles alone when they search for suitable trees on which to oviposit.


Nematology ◽  
2003 ◽  
Vol 5 (6) ◽  
pp. 843-849 ◽  
Author(s):  
Shota Jikumaru ◽  
Katsumi Togashi

Abstract 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.


2020 ◽  
Author(s):  
Hai-Hua Wang ◽  
Can Yin ◽  
Jie Gao ◽  
Ran Tao ◽  
Piao-Piao Dai ◽  
...  

AbstractPine wilt disease (PWD) caused by the nematode Bursaphelenchus xylophilus is a serious problem on pines, and there is currently no effective control strategy for this disease. Although the endoparasitic fungus Esteya vermicola showed great effectiveness in controlling pine wilt disease, the colonization patterns of the host pine tree xylem by this fungus are unknown. To investigate the colonization patterns of pine xylem by this fungus, the species Pinus koraiensis grown in a greenhouse was used as an experimental host tree. The fungal colonization of healthy and wilting pine trees by E. vermicola was quantified using PCR with a TaqMan probe, and a green fluorescence protein (GFP) transformant was used for visualization. The results reported a specific infection approach used by E. vermicola to infect B. xylophilus and specialized fungal parasitic cells in PWN infection. In addition, the inoculated blastospores of E. vermicola germinated and grew inside of healthy pine xylem, although the growth rate was slow. Moreover, E. vermicola extended into the pine xylem following spray inoculation of wounded pine seedling stems, and a significant increase in fungal quantity was observed in response to B. xylophilus invasion. An accelerated extension of E. vermicola colonization was shown in PWN-infected wilting pine trees, due to the immigration of fungal-infected PWNs. Our results provide helpful knowledge about the extension rate of this fungus in healthy and wilting PWN-susceptible pine trees in the biological control of PWD and will contribute to the development of a management method for PWD control in the field.Author summaryPine wilt disease, caused by Bursaphelenchus xylophilus, has infected most pine forests in Asian and European forests and led to enormous losses of forest ecosystem and economy. Esteya vermicola is a bio-control fungus against pinewood nematode, showed excellent control efficient to pine wilt disease in both of greenhouse experiments and field tests. Although this bio-control agent was well known for the management of pine wilt disease, the infection mechanism of fungal infection and colonization of host pine tree are less understand. Here, we use GFP-tagged mutant to investigate the fungal infection to pinewood nematode; additionally, the temporal and spatial dynamics of E. vermicola colonize to pine tree were determined by the TaqMan real-time PCR quantification, as well as the response to pinewood nematode invasion. We found a specific infection approach used by E. vermicola to infect B. xylophilus and specialized fungal parasitic cells in PWN infection. In addition, the fungal germination and extension inside of pine tree xylem after inoculation were revealed. In addition, the quantity of E. vermicola increased as response to pinewood nematode invasion was reported. Our study provides two novel technologies for the visualization and detection of E. vermicola for the future investigations of fungal colonization and its parasitism against pinewood nematode, and the mechanisms of the bio-control process.


2021 ◽  
Vol 61 (4) ◽  
pp. 346-351

The pine wood nematode, Bursaphelenchus xylophilus Steiner & Buhrer 1934 (Nickle 1970) is the major causative agent of the pine wilt disease which has become devastating to Asian and European coniferous forests. These regions are also naturally occupied by two other native but nonpathogenic species, i.e. B. mucronatus Mamiya & Enda 1979 and B. fraudulentus Rühm 1956 which are closely related to the invasive B. xylophilus. Moreover, all these three species can colonize pine trees, and potentially be extracted from the same wood samples. Due to the cosmopolitan character and wide genetic variation within- and between existing populations the taxonomic distinction of these species based exclusively on their morphology is difficult or, almost impossible. The present quarantine regulations related to B. xylophilus require the most credible and simple methods which could allow for a possibly earliest detection and precise identification of this species in wood shipments and conifer forests stands. The main objectives of the presently reported research were to simplify the presently available procedures for possibly fast and precise detection and identification of B. xylophilus examined in the background of the remaining Bursaphelenchus species of the xylophilus group and other bacterio- and mycetophagous nematodes naturally present in the pine wood samples. The developed method is based on a direct examination of the crude nematode extract from wood samples and subsequent use of PCR technique with earlier designed specific reaction starters amplifying ITS1–28S rDNA regions.


Plant Disease ◽  
2021 ◽  
Author(s):  
Yu-Long Li ◽  
Chang-Ji Fan ◽  
Xiao-Hui Jiang ◽  
Xing-Yi Tian ◽  
Zheng-Min Han

Pine wilt disease is the most devastating pine disease caused by Bursaphelenchus xylophilus. Bursaphelenchus mucronatus is morphologically similar to B. xylophilus and geographically overlaps in its distribution. Although interspecific hybridization of the two nematodes has been performed in vitro, the dynamic regularity of hybrid formation and its risk in forests has not been well evaluated. In this study, a hybrid of B. xylophilus and Bursaphelenchus mucronatus mucronatus was identified in the laboratory and fields by molecular markers. The heterozygosity of ITS-5.8S loci for identification was unstable in the hybrid population, and the allele inherited from B. m. mucronatus was lost over several generations. We also provided evidence that hybrids existed in some new epidemic areas, while old epidemic areas were usually dominated by B. xylophilus. Hybrids could be generated when B. m. mucronatus was invaded by B. xylophilus, and the pathogenicity of the hybrids was similar to that of B. xylophilus. These findings may improve the understanding of the natural hybridization between B. xylophilus and B. m. mucronatus and pathogenic variation in pine wilt disease, providing new insights for future studies on disease detection, transmission, and quarantine.


Nematology ◽  
2012 ◽  
Vol 14 (5) ◽  
pp. 547-554 ◽  
Author(s):  
Hayami Kasuga ◽  
Katsumi Togashi

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Ryoji Shinya ◽  
Haru Kirino ◽  
Hironobu Morisaka ◽  
Yuko Takeuchi-Kaneko ◽  
Kazuyoshi Futai ◽  
...  

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world’s most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.


Nematology ◽  
2006 ◽  
Vol 8 (3) ◽  
pp. 435-442 ◽  
Author(s):  
Yuko Takeuchi ◽  
Natsumi Kanzaki ◽  
Kazuyoshi Futai

AbstractPine wilt disease, caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been devastating pine forests in Japan for a century but an effective method for controlling the disease is still needed. Pre-inoculation of the host plant with an avirulent pathogen can induce resistance against subsequent infection with virulent pathogens; this is called 'induced resistance'. Host pine trees have this type of resistance against pine wilt disease but the detailed mechanism is unknown. In this study, 1-year-old potted seedlings of the susceptible Japanese black pine, Pinus thunbergii, were pre-inoculated with an avirulent isolate of PWN (C14-5), as well as an avirulent fungus, Botrytis cinerea, to induce host resistance against PWN. One, 2, 3, 4 and 5 weeks after inoculation, the seedlings were challenged with a virulent isolate of PWN (S10), and the survival of these seedlings was estimated by their external symptoms. Pre-inoculation with either C14-5 or Botrytis delayed the symptom development caused by subsequent inoculation with S10, suggesting that pre-inoculation induced host resistance against PWN. The resistance induced by pre-inoculation with C14-5 lasted for at least 5 weeks, while that induced by Botrytis weakened with time. It has been reported that the PWN survives inside the host seedlings for a long period without causing any symptoms under certain conditions, but Botrytis survives only for a while in the seedlings. Thus, the duration of host resistance induced by pre-inoculation may vary with the viability of the inoculum used for pre-inoculation. In order to obtain durable induced resistance, it may be necessary to repeat inoculations of avirulent microbes to be able to control pine wilt disease.


1988 ◽  
Vol 54 (5) ◽  
pp. 606-615 ◽  
Author(s):  
Keiko KURODA ◽  
Toshihiro YAMADA ◽  
Kazuhiko MINEO ◽  
Hirotada TAMURA

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