Adaptation of pine wood nematode,Bursaphelenchus xylophilus,early in its interaction with two Pinus species that differ in resistance

Pine wilt disease(PWD)is one of the most devastating diseases of Pinu s spp.and is caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner&Buhrer)Nickle.To study adaptation of PWN to survive in hosts that differ in resistance,we examined the self-regulatory characteristics of PWN at the biological and molecular levels early in the interaction.Two-year-old susceptible Pinus thunbergii and resistant Pinus taeda were selected for this experiment,and changes in PWNs after inoculation were assessed.q RT-PCR was used to detect changes in genes related to PWN pathogenicity and detoxification.The results showed that the migration and reproductive abilities of PWNs in P.thunbergii were stronger than those of PWNs in P.taeda.After 7 d,the number of nematodes in P.thunbergii was approximately 3.2-fold higher than in P.taeda.After 15 d,the number of nematodes in P.thunbergii was approximately twofold higher than that in P.taeda.Because PWN can adjust its sex ratio after infection,we compared the sex ratio of uninoculated PWNs,to that in the two pine species.In P.thunbergii,the female to male ratio first decreased and then stabilized over time;in P.taeda first decreased and then increased.Relative fat accumulation in PWNs increased significantly after the PWNs entered the tree body;the accumulation rate in P.thunbergii was higher than in P.taeda at 7 d,but lower after 15 d.Scanning electron microscopy(SEM)showed significantly more bacteria on the surface of PWNs in P.taeda compared with PWNs in susceptible P.thunbergii.At 12 h after inoculation,the expression of genes related to cell-wall degradation(Bx Beta1-4 and Bxpel1),effectors(Bx CDP1,Bx Sap B1),and active oxygen metabolism(Bxy-ctl-1 and Bx GST3)was 2–6×higher in the resistant pine than in the susceptible one.In contrast,in PWNs,the expression of autophagy-related genes Bx ATG1 and Bx ATG16 was 1.5–2 times higher in P.thunbergii than in P.taeda.These results indicate that the interaction between PWNs and pine trees with different resistance levels elicits a series of physiological and molecular adaptations that affect nematode reproduction and virulence.This study will help elucidate the adaptive mechanisms of PWN in different pine trees.

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Linking individual-tree and whole-stand models for forest growth and yield prediction

Background： Different types of growth and yield models provide essential information for making informed decisions on how to manage forests. Whole-stand models often provide well-behaved outputs at the stand level, but lack information on stand structures. Detailed information from individual-tree models and size-class models typically suffers from accumulation of errors. The disaggregation method, in assuming that predictions from a whole-stand model are reliable, partitions these outputs to individual trees. On the other hand, the combination method seeks to improve stand-level predictions from both whole-stand and individual-tree models by combining them. Methods： Data from 100 plots randomly selected from the Southwicte Seed Source Study of Ioblolly pine （Pinus taedo L） were used to evaluate the unadjusted individual-tree model against the disaggregation and combination methods. Results： Compared to the whole-stand model, the combination method did not show improvements in predicting stand attributes in this study. The combination method also did not perform as well as the disaggregation method in tree-level predictions. The disaggregation method provided the best predictions of tree- and stand-level survival and growth. Conclusions： The disaggregation approach provides a link between individual-tree models and whole-stand models, and should be considered as a better alternative to the unadjusted tree model.

Micropropagation of loblolly pine by somatic organogenesis andRAPD analysis of regenerated plantlets

Organogenesis was induced in callus derived from mature zygotic embryos of six families (J-56, S-1003, E-22, E-311, E-440, and Mc) of loblolly pine (Pinus taeda L.) within 24 weeks of culture. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg·L?1 indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). The most suitable medium for root formation proved to be TE medium supplemented with 0.5 mg·L?1 IBA, 2mg·L?1 BA and 0.5 mg/l gibberellic acid (GA3). 169 regenerated plantlets were transferred to a perlite: peatmoss: vermiculite (1∶1∶1) soil mixture, and 98 plantlets survived in the field. Total DNA was extracted from the needles of the regenerated plantlets of the six families of loblolly pine. Analysis of random amplified polymorphic DNA (RAPD) using 20 arbitrary oligonucleotide 10-mers, show that amplification products were monomorphic for all the plantlets of family J-56, S-1003, E-22, E-311, E-440, and Mc of loblolly pine. These results suggested that organogenesis can be used for clonal micropropagation of some families of loblolly pine.

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly pine