Visualization of a host reaction in oak stems infected with a wilt pathogen, Raffaelea quercivora, by magnetic resonance imaging

Keiko Kuroda,1 Yu Ichihara,2 Yoshiyuki Kanbara,3 Takashi Inoue,4 and Akira Ogawa4

1) Kansai Research Center, Forestry and Forest Products Research Institute, Momoyama, Fushimi, Kyoto 612-0855, Japan.
    Email: keiko@affrc.go.jp; HP: http://cse.ffpri.affrc.go.jp/keiko/hp/kuroda-e.html  
2) Tohoku Research Center, Forestry and Forest Products Research Institute, Morioka 020-0123, Japan.
3) High Field Magnetic Resonance Imaging Research Institute, Iwate Medical University, Takizawa-Mura, Iwate 020-0173, Japan.
4) Department of Neurosurgery, Iwate Medical University School of Medicine, Morioka 020-8505, Japan.

     Numerous deciduous oaks, Quercus serrata and Q. crispula, are killed every year in Japan by a fungus, Raffaelea quercivora.  This fungus enters the sapwood vectored by an ambrosia beetle, Platypus quercivorus, which makes mass attacks on healthy oak trees.  Anatomical investigations of infected trees revealed that widespread discoloration (wound heartwood) had occurred in sapwood (Kuroda 2001).  The present report discusses the mechanism of wilting based on the non-invasive observation of infected trees by magnetic resonance (MR) imaging.  This technique is helpful for detecting the water distribution and lesions caused by pathogenic microorganisms in living trees (Kuroda 2005).  
     Q. crispula saplings (diameter: 20-40mm, height: ca. 160cm) planted in pots were inoculated with R. quercivora through one hole or four holes (diameter: 2.5mm) made on the lower stems.  Ten inoculated saplings and five controls were analyzed using an MR imaging system (Signa VH/i 3.0 T; General Electric Medical Systems) at 7- or 10-day intervals for 42 days.  Then, saplings were cut, and the bases were soaked in aqueous acid fuchsin.  Proton density MR images of healthy tree stems showed cambium and functional vessels as high intensity.  In the MR images of the stems inoculated with the pathogen, a darker area around the inoculated holes appeared and was observed to gradually enlarge.  In the specimens inoculated from one hole, the darker areas were observed to elongate about 50mm for six weeks.  Anatomical observations after MR imaging revealed that the darker area in the MR images coincided with the dysfunctional area that had not been dyed with acid fuchsin.  Most of the saplings inoculated with the pathogen through 4 holes started to show leaf discoloration 20 days after inoculation.  Proton density MR images indicated that, prior to leaf discoloration, the dehydrated area had swiftly enlarged horizontally almost covering the stem cross section and longitudinally more than 200mm.　T1-weighted MR images, which are used to detect tissues rich in fat or protein, showed the area of the discolored xylem with fungal distribution as whitish.  MR and optical images demonstrated that the sap flow to the shoots had been blocked at the discolored xylem that had formed as a defense reaction of parenchyma cells to the fungal activity.  Thirty days after inoculation, the MR images showed the accumulation of water in the xylem below the inoculation holes and desiccation above the holes.  Xylem sap oozed from the inoculation holes showing that the root is still functional after the sap at the lower stem had stopped ascending.  These results demonstrated that the swift and wide distribution of the pathogen in sapwood was enhanced by the multipoint infection and that complete cut-off of the water supply to the shoots induced wilt symptom in oak trees.