Received 17 May 2016; accepted 19 July 2016; published 22 July 2016
Indigowoad root (Isatis tinctoria) is one of the most well-konwn approved prescription remedies and is fre- quently used as an anti-leukemia, antipyretic, anti-inflammatory and anti-virus agent  . In addition, a com- pound from indigowoad root granules has been accredited as antiviral agent against influenza virus  . Moreover, the fresh leaves are used as a vegetable. As there is more demand for healthy and nutritional life style, the medial role of indigowoad root will get more attention in future. As an important medical vegetable, the planting area of indigowoad roothassignificantly increased. However, the symptom of the marginal scorch for indigowoad root was observed in the Wuhan City andShennongjia Forestry District, Hubei Province, China (Figure 1(a)). First the margin of leaf became yellow, then gradually crispation and brown. The incidence of symptoms was almost 100%, seriously affecting thecommercial quality of the leaves from indigowoad root. Therefore, the pathogens were isolated by tissue segment method on potato dextrose agarmedium  . A suspension containing 105 conidiophores per ml collected from 7-day-old colonies grown on PDA was sprayed on the foliage of indigowoad root. The control plants were inoculated with sterile water. After inoculation, the plants
Figure 1. The symptom of marginal scorchinfecting indigowoad root. (a) Symptoms in field; (b) symptoms after inoculation.
were placed at 25˚C and 80% humidity. The first lesions appeared after 7 day (Figure 1(b)). Koch’s postulates were fulfilled by consistently reisolating pathogens from inoculated plants, whereas control plants remained healthy. The genomic sequence of 18S rDNA was studied. The DNAs of mycelia were extracted with CTAB method  . The sequences of primers were designed as follows: 18S-1,5’-GTAGTCATATGCTTGTCTC-3’; 18S-2, 5’-TCCGCAGG TTCACCTACGGA-3’. The PCR reaction conditions were as follows: The settings for the thermal profile included an initial denaturing at 94˚C for 2 min, followed by 25 cycles of amplification (94˚C for 30 s; 45˚C for 30 s; and 72˚C for 2 min) and a finally extension at 72˚C for 5 min. The PCR products were detected by 1% agarose gel electrophoresis. A band was detected around 1700 bp. The amplification products were ligated, transformed and sequenced. The results were aligned with the sequences in GenBank. The 18S rDNA sequence of the representative pathogen isolated from the fresh leaves of the indigowoad root was deposited in GenBank (accession no. KU512834). The basic local alignment search tool (BLAST) was used to indicate functional and evolutionary relationships between sequences, identifying members of gene families. BLAST search of this nucleotide sequence illustrated 99% identity with 18S rDNA sequences of several Cladosporium sp. isolates available in GenBank. So the species was identified as Cladosporium sp. This is the first reported Cladosporium sp. infection in fresh leaves of the indigowoad rootin China, and we believe that this information will be useful for studying Cladosporium sp. infection in other vegetables. At the same time, our results indicate the need for the revision of indigowoad rootmanagement.
This work was financially supported by the Modern Agricultural Industry Technology System Project Supported by Ministry of Agriculture (nycytx-35-02-06).
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