Study of White Root Rot Disease of Camellia sinensis in Taiwan

Eleven isolates, appearing the typical hyphal characteristic of septa adjacent pear-shape swellings of Rosellinia necatrix Hartig, were isolated from white root rot samples of Camellia sinensis (L.) O. Kuntze, gathered from five tea gardens that suffered from the dieback disease in mountainous Nantou during the study. The isolation rate was 2.41 % for tea samples and 5.38 % for tea gardens. In addition, 25 pathogen isolates originated from other hosts were also collected from the central and northern areas of Taiwan. 48 ascospore isolates were also collected from 7 mature ascomata produced by isolate R203. The synnemata (anamorph) produced by the field isolates were black and bristle-like, tapering toward the enlarged whitish gray heads. Conidium was hyaline, one-celled, subglobal, and base truncated, 2.9-5.8 × 1.9-3.5 μm. The ascoma was single or gregarious, dark brown to black, uniperitheciate, subglobose to globose. Ascus was unitunicate, clavate to cylindrical, contained 8 uniseriate ascospores, with a basally conical, apically enlarged and strong blue amyloid apical apparatus. Ascospore was one-celled, brown to dark brown, asymmetrically narrow fusiform with two acute ends, 37.0-55.0 × 5.4-7.9 μm, with a less than a half, short straight germ slit parallel to its longitude. Based on the result of ITS DNA sequencing comparison and phylogenetic analysis, 36 isolates of white root rot pathogens collected from Taiwan except R701 were identified as Rosellinia necatrix Berl. Prill. Isolate R701, which was relatively more polymorphic in ITS DNA sequence than others but still had high similarity with the consensus sequence (over 97 % similarity), was temporarily regarded as R. necatrix related pathogenic Rosellinia sp. The ITS DNA sequences of isolates R101, R203, R210, R301, R401, R701 and R801, have been deposited in NABI GenBank data library. The optimum temperature for radial growth of R. necatrix mycelium was 25 ℃. No growth was observed when temperature over 35 ℃. On the contrary, there was still 0.1 mm mycelial growth when incubated at 5 ℃ after 7 days. The optimum pH for radial growth was around pH 5.0. Glucose was the best carbon source to enhance the radial growth of the pathogen, while xylose was the inferior one. The organic nitrogen sources, peptone and casein, could enhance the mycelial growth of the pathogen, while inorganic nitrogen sources such as calcium nitrate or urea seemed to retard radial growth. The VCGs of white root rot pathogen, 18 vegetative compatible groups existing among 36 field isolates, were quite complex. All the 14 isolates from Acer morrisonense belonged to VCG1, isolate R202 and R204 from Serissa japonica belonged to VCG2, and isolate R203, R205, R206 and R208 from S. japonica and isolate R209 from Ehretia microphylla belonged to VCG3. The other field isolates and ascospore isolates were mycelial incompatible with each other, even the ascospore isolates and the parental isolate R203 were mycelial incompatible. The result that isolates within the same VCG were genetically undistinguished by AFLP analysis indicated they were originating from a genet. AFLP analysis also showed that 50 % ascospore isolates were highly genetically related to parental isolate R203. It demonstrated that AFLP analysis could suitably be applied to explore the genetic structures and trace the dispersal of R. necatrix. When artificially inoculated to tea cuttings, the pathogen isolates, which could be re-isolated from dead infected tea cuttings to fulfill Koch’s postulates, produced tea white root rot symptoms similar to those observed in diseased fields. Pathogenicity determination indicated that all the field isolates including R701 and ascospore isolates were highly virulent except those originated from A. morrisonense. Therefore, it suggested the possible ways of dissemination of white root rot disease in Taiwan including mycelial strands transmittion as well as ascospores dispersal. The EC50s of azoxystrobin, pyraclostrobin, carbendazim, prochloraz, and fluazinam against the pathogen were quite sensitive at the ppb level. Drug dipping tests for the previous chemicals against inoculated twigs showed no statistically significant difference (α=0.05) in inhibitive effects. The result of chemical drenching for tea white root rot disease in greenhouse demonstrated that the control effect of the previous 5 chemicals at 1000X dilution had no statistically significant difference (α=0.05). No harmful drug side-effects were observed for tea cuttings of treatments, whereas those of disease control died within 4 months. Therefore, azoxystrobin, pyraclostrobin, carbendazim, prochloraz, fluazinam could be taken into consideration for chemical control of white root rot disease of Camellia sinensis in the future.