Quick-Detection of Brown Root Rot Disease in Tree

106

Brown root rot disease ( Brr. ) is a tree disease caused by Phellinus noxius in warm and humid areas, including Taiwan. It infects hundreds species of tree plants, often causing tree wood decay, death of living trees, and tree falling all around Taiwan island. Brown root rot disease usually spreads by the underground root-to-root contact, human dissemination of the diseased tissue or seedlings, or the infestation by diseased debris. The basidiocarp and basidiospores may also play roles for long distance dissemination although very rare. At present, the detection of Brr can be classified into the following five categories: Detection or diagnosed by symptoms or signs, detection by its specific DNA sequence, detection by its immunological antigen, detection by the emitted VOC (Volatile organic compounds), and detection by physiological or biochemical characteristics. The current nine practical detection methods include the symptoms and signs diagnosis or called VTA (Visual tree assessment) method, microscopic examination, polymerase chain reaction (PCR) method, LAMP( Loopmediated isothermal amplification )method, ELISA (Enzyme-linked immunosorbent assay), electronic nose method, detective dog method, selective medium method, and histological staining method. In this study the SWOT（ Strengths, weaknesses, opportunities, threats） of these nine methods are analyzed based on criteria such as scale of the detection target, required time, specificity, cost, easiness, and ability to detect the live mycelium, Results showed that only selective medium method, microscopic examination, and histological staining have the ability to detect the live mycelium, and are important for Brr detection after tree injection or soil disinfection. For detecting larger targets, the symptoms and signs diagnosis, electronic nose method, and detective dog method are appropriate. For quick Brr detection, the symptoms and signs diagnosis, microscopic examination, histological staining, and detective dog method are useful. For higher specificity, the PCR, LAMP, and ELISA, are the choices. For lower cost, the symptoms and signs diagnosis, and microscopic examination are suitable. For easiness in the field, the symptoms and signs diagnosis, microscopic examination, LAPM method, electronic nose method, and histological staining, can do the jobs. In this study the previously published selective medium (MA+4), for isolating Brr pathogen, was modified by doubling the benomyl concentration. This named PN3 medium can prevent the interference caused by Trichoderma and Penicillium spp. Then the basal nutrient components of PN3 was modified by replacing the malt extract (ME) with 1/2 ME + 1/2 PDA, named as PDM, for improving the dark patch development of Brr pathogen on PDM directly. Finally new inhibiting biocides such as fosetyl-aluminium, kasugamycin copper, thiophanate-methyl, and tetracycline with streptomycin, were added into PDM+4, making new formula as PDM+8. This PDM+8 selective medium shows higher selective power against the bacteria and other wood rot fungi, increasing its specificity and easiness for detecting the Brr pathogns. The new histological staining methods were also tested out in this study. The Brr mycelium on PN3 or PDM+8 for 2 to 3 days expressed the stunt head and identical branch width, that were specific characteristics of the pathogen. New stains or dyes such as methyl blue, crystal violet, Malachite green, Safranin O, were tested to stain the mycelium within the field tree samples. Results showed that the cotton blue for Brr pathogen performed the best for histological staining jobs. In the cultural medium the Brr pathogen showed two distinguishable hyphae. One is the skeleton hypha which is not stained by cotton blue. Another is the common hypha or arthrospore that can be stained by cotton blue. As the colony become mature or older, some common mycelium become porous or empty within the mycelium, named as porous mycelium, which is a specific characteristic for Brr pathogen. Using the cotton blue to stain the Brr in field tree samples, both skeleton hypha and porous mycelium can be observed under the microscope, making it an easy method for detecting the Brr in the field or in the laboratory.