**2.4 The pine wilt disease in China**

In mainland China, the pine wilt disease was reported for the first time in a small patch of *Pinus thunbergii* near the Sun Yat-sen Mausoleum in Nanjing, Jiangsu Province in 1982 (Y.Y. Wang et al., 1991). Afterwards, the infested area expanded quickly throughout eastern China. From the bulletins released by the SFA (State Forestry Administration) of China, a clear tendency of westward expansion can be observed (SFA, 2004 ~ 2007). During the past two years, some counties in Henan, Shaanxi, and Sichuan provinces in northern, northwestern, and southwestern China also reported infested areas of the pine wilt disease (SFA, 2011) (Fig. 1).

Detecting Non-Local Japanese Pine Sawyers

**4. Validating the hypothesis – Theory and experiment** 

research on *M. alternatus* in China published by the end of 2009.

with barkborer lure (Chinese Academy of Forestry, Zhejiang, China).

**4.1 Molecular achievements on** *Monochamus*

from the suspected region.

**4.2 Designing the experiment** 

marker.

in Yunnan, Southwestern China via Modern Molecular Techniques 73

constructing telecommunication facility, which consumed a considerable amount of electronics manufactured in eastern China (Z.Q. Li, pers. comm.). Hence, a reasonable hypothesis was conceived that the nematodes in Wanding were casually introduced with non-local *M. alternatus* populations hidden in the wood-packaging materials. Providing that *M. alternatus* from different localities possess different genetic profiles, the non-local individuals can then be distinguished by analyzing the genetic differences on population level, and the source can be traced by comparing the genetic profiles with the samples taken

Although *Monochamus* species are important forest pests across the Eurasia, research employing molecular techniques were quite limited compared to other arthropod pests. Cesari et al. (2005) started the first molecular taxonomy research on seven *Monochamus* species by using the combined mtDNA data of *cox1* and *12s* genes. Afterward, such research were quickly developed in Japan, where *M. alternatus* had become a threat to forestry. Kawai et al. (2006) published a paper on the genetic structure of 27 populations of *M. alternatus* from Japan and China (with 25 populations from Japan and two populations from China). Shoda-Kagaya (2007) published paper on microsatellite markers. Then, Koutroumpa et al. (2009) conducted a criticizing research which revealed *Numts* resembling the *cox1* and *cox2* genes of *M. galloprovincialis* and *M. sutor*, alerting scientists that precautions are necessary when applying the gene markers from the mitogenome. There was no such

Appropriate sampling strategy requires samples to be taken from populations scattered in the entire research range, and for each population, sufficient individuals should be collected to represent it (S.Y. Chen & Y.P. Zhang, 2006). Based on this, seven populations representing *M. alternatus* from central, southern, southwestern, and northern Yunnan were selected, in which three populations were chosen from the southwestern Yunnan where the pine wood nematodes were reported. In an attempt to determine the source of the pine wood nematode, two reference populations from outside Yunnan were set, with one from Hubei Province representing central China and the other from Zhejiang Province representing eastern China (Fu et al., 2010; Fig. 2). Adult *M. alternatus* were collected by flight traps baited

Choosing the correct genetic marker can minimize problems in data analysis (Simon et al., 1994) and maximize the future potential of cross references with data published by other researchers. Population phylogeny requires gene markers with moderate evolutionary rate, free of recombination, which means that mtDNA, a maternal heritage genome without recombination, is the ideal choice. Considering that there has been no report on *Numts* in *M. alternatus*, which can also be detected and excluded by strict data examination when encountered. The prefunding research by Kawai et al. (2006) resolved a clear population phylogeny using a gene fragment of *cox2*; the research discussed here chose the same *cox2*

Fig. 1. The distribution pattern of the pine wilt disease (red dots) in mainland China based on the historical data from 2004 ~ 2011, data source: SFA (2004 ~ 2011).

With the expansion of pine wilt disease, an interesting distribution pattern was revealed. In eastern and southern China, the distribution concentrated near harbors, like the deltas of the Yangtze River and the Pearl River, while in the central portion of China, the infested areas are mostly along the Yangtze River. Such a distribution pattern suggested that the dispersal of *B. xylophilus* was closely related to transportation.

The occurrence of the pine wilt disease in Yunnan Province, southwestern China was distinct. Not only because it was one of the latest infested areas after the massive expansion, but also due to its unique geographical position. Unlike eastern China where many harbors are distributed, the first, single, and isolated pine stand (*Pinus kesiya* var. *langbianensis*) in Yunnan infested by *B. xylophilus* was located in the westernmost corner, a remote township named Wanding near the Sino-Burmese border (SFA, 2007).
