Studies On The Pathogens Of Pine Wilt Disease And The Taxonomic Status Of A French Isolate

Pine Wilt Disease (PWD), the pathogen of which is Pine Wood Nematode (PWN) [Bursaphelenchus xylophilus (Steiner&Buhrer) Nickle] in extensively accepted view, has been a devastating disease in forestry since the last century. However, in addition to PWN, a morphologically extremely similar species B. mticronatus Mamiya&Enda is suspected as possessing pathogenic potential to pines. More complicatedly, accompanying bacteria has been added among the focus candidates as the possible pathogen of PWD in recent years. In addition, a French isolate is special in pathogenicity, so some experiments must be performed to clarify its taxonomic status. To resolve all the above questions and acquired more data for further investigation, the following experiments were carried out.Twenty-seven populations of B.mucronatus were inoculated to 3-year-old seedlings of Pinus massoniana and P. thunbergii for evaluation of their pathogenicity. The results showed that P. thunbergii seedlings demonstrated much higher susceptibility while no obvious symptoms were found in P. massoniana seedlings after inoculation. Further data analysis agreed unanimously with the symptom observation since to Pinus massoniana seedlings, the mortality rate and RHS index of B.mucronatus are both 0, and to P. thunbergii seedlings, mortality rate ranged between 0 and 75%, RHS index between 0 and 840. Especially, a population, with 75% mortality and 840 value of RHS index to P. thunbergii seedlings, displays equivalent pathogenicity against PWN. Attentions also need to be paid on another two populations BmFr2 and Bmcan due to their same mortality rate 50% and respective values of RHS index 207.2 and 117.2. BmCWY1, BmCGHP, BmCGHH, BmCSX and BmFr1, all with RHS values below 100 and yet partial mortality to P. thunbergii seedlings, fell into one group. However, it's clear that most populations of B.mucronatus put no effect on seedlings of both P. massoniana and P. thunbergii.To confirm the effects of so-called accompanying bacteria on pine wilt, 4 strains of bacteria were isolated from the body surface of 4 populations of PWN and used in inoculation test. The results discovered that un-sterile nematodes, sterile nematodes and the mixture of sterile nematodes plus their corresponding bacteria all caused 100% mortality to 3-year-old P. thunbergii seedlings. Individual bacteria without nematodes led to no death ofinoculated seedlings, so the mortality rate is 0. t test to RHS values showed: (1) On the 0.01 significance level, it's significant different between un-sterile nematodes and sterile nematodes, the mixture of sterile nematodes plus their accompanying bacteria; (2) On the 0.05 significance level, between sterile nematodes and the mixture of sterile nematodes plus their accompanying bacteria. All these data supported that bacteria contributed to the suitability of nematodes inside their host pines.By means of morphological comparison, molecular identification using ITS-RFLP patterns and biological hybridization, 2 populations of PWN BxUS3, BxCAJ, 3 ones of B.mucronatus with asia-genotype BmFJ2, BmCNJ, BmCSC1, the above-mentioned isolate BmFr2 and two unpublished new species Bnspl and Bnsp2 were investigated to clarify the taxonomic status of the French isolate. The results showed that the morphological characters and the ITS-RFLP pattern of BmFr2 are more similar to those of B. mucronatus with Asia-genotype rather than B. xylophilus. Also, the successful hybridization between BmFr2 and BmFJ2, BmCNJ, BmCSCl, not between BmFr2 and BxUSS, BxCAJ, Bnspl, Bnsp2 means that BmFr2 doesn't belong to a separate species but should be B.mucronatus. However, determining the taxonomic status of French B. mucronatus need further investigation.