Comments on Bursaphelenchus mucronatus provided by Ke Dong and the nematologists of the California Department of Food and Agriculture (CDFA).

The pine wood nematodes, Bursaphelenchus xylophilus and B. mucronatus, are members of the pinewood nematode species complex (PWNSC). Biological studies showed that these two nematodes shared strong similarities in many respects – morphologically, vector relationships, life cycle and host trees. The tail of Bursaphelenchus xylophilus females is hemispherical with a smooth rounded terminus, whereas the tail of Bursaphelenchus mucronatus females is mucronate as shown in the following figure:
B. xylophilus tail
B. mucronatus tail

Both B. xylophilus and B. mucronatus females are provided with a vulval flap shown in the following figure:

B. xylophilus is a native species to North America. It was first found in association with fungi in timber in 1929 and described as Aphelenchoides xylophilus (Steiner and Buhrer, 1934). Nickle (1970) transferred this nematode to Bursaphelenchus (JON 2:375-392). Mamiya and Kiyohara (1972. Nematologica 18:120-124) reported Bursaphelenchus lignicolus (n. sp.) in Japan but this species was placed as a synonym of B. xylophilus (Nickle, 1981. JON 13: 385-392). It was believed that the nematode was introduced from North America to Japan around the turn of the century. Molecular data also supported the theory that the Japanese and American strains of B. xylophilus might be derived from common origins. It was first demonstrated to be a pathogen on pine tree in 1971 (Kiyohara and Tokushige). The wilting caused by B. xylophilus in susceptible pines (Asian species) results in serious economic and environmental damage to Chinese and Japanese pine forests. Because this disease is regarded as a serious threat to European forests, the European Community (EC) has instituted a ban on the import of coniferous timber which has not been kiln dried from regions where B. xylophilus is endemic. In the US, however, B. xyluphilus has not shown any epidemic wilting disease in American pine species. In addition, the nematode has not caused the death of American pine species planted in China and Japan, suggesting that North America pine species may have resistance to this nematode.

Bursaphelenchus mucronatus (Mamiya and Enda, 1979, Nematologica 25: 353-361) is widely distributed in Eurasia, from most of the European countries to Japan. B. mucronatus has been recorded in Canada, but has not been found in the United States.

B. mucronatus was originally ranked as a “M” in the list before the working group started to solicit input from SON members. There were two comments received from the SONLIST, both seemed to be negative. Dr. McNamara (Assistant Director, European and Mediterranean Plant Protection Organization, Paris, France) said that the importance of this nematode as a pest had not been clearly established. B. mucronatus had never been shown to cause the death of trees under nature conditions in Europe. Dr. Dwinell (US Forest Service, Athens, GA) mentioned that some North America pine species were introduced and grown in those countries where B. mucronatus was common, and there had been no report of these pines being killed by the nematode. His conclusion was that: “B. mocronatus is not a pathogen of pines”.

Having reviewed the available information from the published literatures, B. mucronatus has been reported as a weak pathogen. B. mucronatus is often found in dead or dying pine trees but not known to initiate disease in nature in non-stressed trees. Inoculation test showed that the mortality caused by B. mucronatus was low (4.2%-6.2% in general), which was not much greater than non-inoculated controls. Under the same testing conditions, however, B. xylophilus could cause greater than 64 percent tree death. No typical wilting symptoms in pine trees infected by this nematode are reported in nature in either Chinese or Japanese publications (Mamiya and Enda, 1979, Nematologica 25: 353-361. Cheng, et. al., 1986. Journal of Nanjing Agric. Univ. 1986: 55-61).

B. mucronatus is reported to have a broader distribution than B. xylophilus in Asia. Biological studies have shown that these two species can be isolated from the same infected hosts or dead trees, and they share the same insect vectors in nature. ‘Pure’ cultures can be established under the lab conditions by selecting and inoculating with singe adult females. The two species are differentiated by the shape of the tails (Cheng et. al. 1986). B. mucronatus differs morphologically from B. xylophilus only by the presence and length of a mucro (>3.5 µm) on the tail terminus. Certain isolates of B. xyluphilus are also reported to have mucronate tail but usually the mucrones are not longer that 2 µm.  Similarly in North America, morphotypes of B. xylophilus with some characteristics of B. mucronatus have been recovered in the US and Canada, they are called “M” forms (mucronate form) as compared to typical “R” form (round tail).  Molecular techniques have been conducted to differentiate the two species and certain DNA fragments may have value in determining taxonomic affinities (Harmey and Harmey, 1993, JON25: 406-415). ITS sequences also support the perception that B. mucronatus group and B. xylophilus are distinct genetic entities (Becknbach et. al., 1999, Nematology 1: 539-548). Based on the molecular data (Harmey and Harmey, 1993, JON25: 406-415. Beckenbach et. al., 1992. JON24: 140-147), there exist two distinct B. mucronatus groups: one group containing the European isolates and another containing the Japanese isolates. In addition, tremendous DNA-based variations are found among the isolates of PWNSC, at both species and strain levels.

Differences among virulent and avirulent isolates of B. xylophilus have been reported in Japan and in the US. In general, the “R” form isolates showed higher virulence to tested pine species, e.g. Pinus sylvestris, P. strobus, P. nigra, P. taeda and P. thunbergii. However there were also some avirulent “R” forms, or the pine species showed strong tolerance to these isolates (Bolla and Boschert, 1993. JON 25: 227-238). Among the “M” forms, a Japanses isolate was highly virulant to the tested pine trees, but the other isolates were moderately virulence (Bolla and Boschert, 1993). An “M” form of B. xylophilus was reported to be highly virulent on Pinus massoniana in china (Cheng, 1986). “M” forms also have been recovered from dead or dying conifers in France, Norway and Siberia. A French “M” form isolate that was found in declining Pinus pinaster trees showed more pathogenic than B. mucronatus, but not as pathogenic as B. xylophilus (de Guiran and Boulbria, 1985. Nematologica 35: 321-330). There were two B. mucronatus isolates tested in Bolla’s report (1993), both showed avirulent to the tested pine species.

The biological species concept is based on the definition of interbreeding. The validity of B. mucronatus was contested by Baujard (1980), but confirmed by Nickle et al (1981) on the basis of its reproductive isolation from B. xylophilus in mating experiments (Yashuaru Mamiya’s test being mentioned in Nickle’s report, JON13: 385-392). However, inter-specific hybridization has been demonstrated between B. mucronatus and B. xylophilus in many later publications. de Guiran and Bruguier (1989) reported that a French “M” form isolate could produce normal fertile hybrids with B. mucronatus (a Japanese isolate) as well as with B. xylophilus (also a Japanese isolate), but the same isolate did not produce fertile hybrids when mated with an “M” form isolate from Minnesota (Nematologica 35:321-330). Riga et al. (1992) conducted an interspecific and intraspecific cross hybridization test, in which the male nematodes from a Japanese B. mucronatus isolate mated successfully with females of four different isolates of B. xylophilus, suggesting that the two species came from a common ancestor (Fundam. Appl. Nematol. 15: 391-395). Bolla and Boschert (1993) demonstrated that interbreeding occurred in the laboratory between some “M” and “R” forms of B. xylophilus, and the hybrids exhibited the pathogenicity of the parent with the broader host range. Interbreeding of B. xylophilus and B. mucronatus was rare but did occur. They suggested that virulence might be inherited as a dominant character or that increased virulence might have resulted from differences in hybrid vigor (JON 25: 227-238).
 

Considerations for keeping B. mucronatus in the SON list:

It is not known to occur in the US.

It is a weak pathogen to some pine species, but inter- and intra-specific variations among isolates of nematodes are observed, and the nematode strain/pine host specific interactions are not thoroughly understood.

The nematode is not known to be a killer to pine trees, but the decline and growth reduction caused by the infection is not clear.

Susceptibility of US pine species under the nature conditions in North America is unknown. Variations of susceptibilities may exist at strains, varieties, sub-species and species levels.

Interbreeding with B. xylophilus has been demonstrated. The hybrids may have the pathogenicity of the parent with the broader host range, and interbreeding of virulent isolates with avirulant isolates might lead to production of new, highly virulent hybrids.

Morphological identification is not always reliable. Dwinell and Nickle considered “M” form isolates to be closely related to B. mucronatus, but molecular data indicated that the “M” forms were within the B. xylophilus group. Unequivocal identification of these nematodes is further complicated by the inter-fertility of B.mucronatus and B. xylophilus. Many nematologists preferred to use the terms such as the pine wood nematode species complex (PWNSC) or supra-species to address this group of nematodes.

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Other authors such as Dave Dwinell and Robin Giblin-Davis don’t share the concern of the nematologists of the California Department of Food and Agriculture

Dave Dwinell states that the data presented in the previous document don’t change his position. He considers this nematode a low risk pest that should be removed from the list. In his view, the ability of B. mucronatus to interbreed with B. xylophilus has little significance, since these two species co-exist only in Japan.

Robin Giblin-Davis provides useful comments about the risk status of B. mucronatus.  He stated:
‘Pine pathology appears to represent a recent invention (apomorphy) that helps separate the closely related species B. xylophilus from B. mucronatus and the other closely related species of that clade, such as B. conicaudatus and B. fraudulentus. Both species produce cellulases as I recall, but B. xylophilus is more mobile which may cause some of the problems. However, even B. seani, a soil inhabitant and fungal feeder, which has nothing to do with plants can be cultured on alfalfa callus (see one of my old papers, ca 1983). Thus, cellulases may be relatively common (pleisomorphy) when we start looking in the Aphelenchoididae. B. xylophilus from B. mucronatus are part of a clade within the Bursaphelenchus that includes B. cojicaudatus (Kanzaki et al. 1998-2002) and B. fraudulentus. When the hosts are all known for this group, I would guess that they will be mostly cerambycid longhorn beetles and the nematodes are fungal feeders like the stem ancestor for the genus.
The problem comes in guessing about the potential pathogenicity of B. mucronatus or any of the 55-60 described species (potentially 100-1000s more) to a country of non-origin or to non-endemic plants. Anything that we say will be a guess. I am reminded of the movie "Finding Nemo" when the clown fish, Marlin says to the blue fish, Dori, "How do you know that something bad isn’t going to happen?" Dori replies, "You don’t". That is the paradox in regulatory decisions….You do not want bad things to happen if they could be prevented by relatively simple exclusionary measures. However, the risk is to become paralyzed into excluding everything.
I agree with Dr. Dwinell, that if you put B. mucronatus on the list without any significant pathogenecity data (and in my mind there isn’t much…injection studies are not good for real world extrapolations and non-native (North American) pine species are not exhibiting pathology in B. mucronatus areas in Europe and elsewhere) then you will have to put all species of Bursaphelenchus on the list (and Aphelenchoides) associated with pines because you do not know what will happen until it happens. From our data, B. cocophilus looks like it evolved to be a strong plant parasite out a different clade within Bursaphelenchus. Thus, given time, perhaps any aphelenchoidid could become a potential problem in the right situation. This takes the argument to the extreme but perhaps it illustrates the point that we are not very good at predicting what organisms will become a problem.
I would argue not to put B. mucronatus on the list. However, I would suggest that a watch be put on it and that more extensive real world pathogenicity testing be done. You should also understand that "B. mucronatus" appears to involve two "species" that would need to be evaluated. I hope that this helps’.

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After a serious evaluation of the comments reported above, the Working Group decided to leave B. mucronatus on the list but with a low (L) ranking status.