China is the world's largest producer country of coptis (Coptis chinensis), the rhizomes of which are used in traditional Chinese medicine. Since 2008, however, root rot symptoms, including severe necrosis and wilting, have been observed on coptis plants in Chongqing, southwestern China. Of the plants examined from March 2011 to May 2013 in 27 fields, 15 to 30% were covered with black necrotic lesions. The leaves of infected plants showed wilt, necrotic lesions, drying, and death. The fibrous roots, storage roots, and rhizomes exhibited brown discoloration and progressive necrosis that caused mortality of the infected plants. Infected plants were analyzed to identify the causal organism. Discoloration of the internal vascular and cortical tissues of the rhizomes and taproots was also evident. Symptomatic taproots of the diseased coptis were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed in sterile distilled water for 2 min, and then air-dried in sterilized atmosphere/laminar flow. Small pieces of disinfested tissue (0.3 cm in length) were transferred to petri dishes containing potato dextrose agar (PDA) supplemented with 125 μg ml–1 streptomycin sulfate and 100 μg ml–1 ampicillin, and incubated for 5 days at 25°C with a 12-h photoperiod. Four distinct species of fungal isolates (HL1 to 4) derived from single spores were isolated from 30 plants with root rot symptoms collected from the study sites. To verify the pathogenicity of individual isolates, healthy coptis plants were inoculated by dipping roots into a conidial suspension (106 conidia/ml) for 30 min (15 plants per isolate), as described previously (1). Inoculated plants were potted in a mixture of sterilized quartz sand-vermiculite-perlite (4:2:1, v/v) and incubated at 25/18°C and 85 to 90% relative humidity (day/night) in a growth chamber with a daily 16-h photoperiod of fluorescent light. Plants dipped in sterile distilled water were used as controls. After 15 days, symptoms similar to those observed in the field were observed on all plants (n = 15) that were inoculated with HL1, but symptoms were not observed on plants inoculated with HL2, HL3, and HL4, nor on control plants. HL1 was re-isolated from symptomatic plants but not from any other plants. Morphological characterization of HL1 was performed by microscopic examination. The septate hyphae, blunt microconidia (2 to 3 septa) in the foot cell and slightly curved microconidia in the apical cell, and chlamydospores were consistent with descriptions of Fusarium solani (2). The pathogen was confirmed to be F. solani by amplification and sequencing of the ribosomal DNA internal transcribed spacer (rDNA-ITS) using the universal primer pair ITS4 and ITS5. Sequencing of the PCR product revealed a 99 to 100% similarity with the ITS sequences of F. solani in GenBank (JQ724444.1 and EU273504.1). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the HL1 isolate in a well-supported cluster (97% bootstrap value based on 1,000 replicates) with JQ724444.1 and EU273504.1. The pathogen was thus identified as F. solani based on its morphological and molecular characteristics. To our knowledge, this is the first report of root rot of coptis caused by F. solani in the world. References: (1) K. Dobinson et al. Can. J. Plant Pathol. 18:55, 1996. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006.
In-vitro compatibility assay of indigenous Trichoderma and Pseudomonas species and their antagonistic activities against black root rot disease (Fusarium solani) of faba bean (Vicia faba L.)
