Web blight ( Thanatephorus cucumeris ): a new disease on leaves of okra plants Mancha areolada ( Thanatephorus cucumeris ): nova doença das folhas do quiabeiro

: In an experiment on organic production of okra ( Abelmoschus esculentus (L.) Moench) that was carried out from September 2013 to January 2014, in Manaus, Amazonas state, Brazil, we observed large chlorotic, necrotic, helical, discontinuous, dark or light-brown lesions with partial detachment of the injured area on the adaxial surface of leaves located in the median and basal portions of the plants. A whitish mycelium mantle covers the lesions on the leaves at the abaxial surface at high moisture conditions. Using morphological characteristics, Koch’s postulates, and phylogenetic analyses of the ITS-5.8S rDNA region, we identi fi ed that the fungus causing the lesions on the okra leaves was Th anatephorus cucumeris (Frank) Donk (asexual stage of Rhizoctonia solani Kuhn of the anastomosis group AG-1 ID). Th is is the fi rst report of T. cucumeris causing web blight on okra in Brazil, and probably in the world. So far, T. cucumeris was described on okra only on post-harvest pods rotting and seedlings’ damping o ff .


ABSTRACT:
In an experiment on organic production of okra (Abelmoschus esculentus (L.) Moench) that was carried out from September 2013 to January 2014, in Manaus, Amazonas state, Brazil, we observed large chlorotic, necrotic, helical, discontinuous, dark or light-brown lesions with partial detachment of the injured area on the adaxial surface of leaves located in the median and basal portions of the plants.A whitish mycelium mantle covers the lesions on the leaves at the abaxial surface at high moisture conditions.Using morphological characteristics, Koch's postulates, and phylogenetic analyses of the ITS-5.8SrDNA region, we identified that the fungus causing the lesions on the okra leaves was Thanatephorus cucumeris (Frank) Donk (asexual stage of Rhizoctonia solani Kuhn of the anastomosis group AG-1 ID).This is the first report of T. cucumeris causing web blight on okra in Brazil, and probably in the world.So far, T. cucumeris was described on okra only on post-harvest pods rotting and seedlings' damping off.

PALAVRAS-CHAVE:
In an experiment on organic production of okra carried out in an experimental area at Embrapa Amazônia Ocidental, Manaus, Amazon state, Brazil, from September 2013 to January 2014, we observed large chlorotic, necrotic, helical, discontinuous, dark or light-brown lesions with partial detachment of the injured area on the adaxial surface of leaves located in the median and basal portions of the plants (Fig. 1).An easily seen whitish mycelium mantle covered the spots on leaves of the abaxial surface at high moisture conditions, which covered the whole colonized area (Fig. 2).
The fungus was isolated using a potato dextrose agar (PDA) medium.The pathogenicity test was made under greenhouse conditions.The mycelium found in the abaxial surface of young okra leaves inoculated the PDA blocks.After the inoculation process, the plants were kept in a humidity chamber for 24 hours.We identified the pathogen based on morphological characteristics, Koch's postulates, and on the ITS-5.8SrDNA sequence region of fungus.
We observed the morphological characteristics in the asexual phase culture grown on the PDA medium and on the basidiospores, by adapting the technique used by TRINDADE et al. (1983) to produce these spores.We used distilled water to wash parts of leaves showing fully developed lesions and containing the pathogen's mycelium on the abaxial surface collected at the field.Then, we took 2-cm 2 pieces of the lesioned parts of the leaves.Each piece was placed at the inner side of the lid of a Petri dish along with a cotton sliver, and secured using adhesive tape.The leaf 's abaxial surface faced the bottom of the dish.After moistening the cotton sliver using sterile water, we placed the lid onto the Petri dish that contained the PDA medium.After preparation, the Petri dishes were incubated at 23ºC under environmental lighting at the laboratory.Under these conditions, we noticed the ejection of basidiospores after two hours.
PDA-grown hyphae, at their asexual phase, showed wideangle branches (nearly 90º) featuring a small constriction at their point of origin, which is a characteristic of Rhizoctonia species.On the pieces of leaves containing the pathogen's mycelium that were placed in the humidity chamber, the hymenium's hyphae produced 6 to 18 µm diameter and 9 to 26 µm height basidia -from barrel-shaped to cylindrical basidia -arranged individually or in arrays similar to clusters.Each basidium produced an average of four sterigmata (varying from 3 to 7), which measured from 35 to 53 µm in length.The ejected basidiospores were hyaline, oblong, with a thin and flat wall, and measured 6-13 µm × 4-9 µm.
The pathogenicity test showed symptoms and signs of the pathogen in the third and fifth days after inoculation, respectively.The pathogen was then re-isolated in the PDA medium for confirmation of Koch's postulates.
To obtain the pathogen's phylogenetic position, we determined the rDNA ITS-5.8Ssequence region of the fungus.The fungus' lyophilized mycelial DNA was extracted using the Genelute kit (Sigma-Aldrich Brasil), and following the manufacturer's instructions.The DNA extraction procedure was performed in triplicate.To amplify the polymerase chain reaction (PCR) and the rDNA's ITS region sequencing, we applied the ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGT CGTAACAAGG-3') pair of primers (WHITE et al., 1990).
The PCR products were sent to Macrogen ® (Korea) and submitted to a sequencing reaction using the PE Applied Biosystems ABI-3700 automatic sequencer.We analyzed the sequences using the Geneious software (Biomatters Limited,  Table 1.DNA sequences of rDNA's ITS1-5.8S-ITS2region from standard isolates of AG-1 and AG-4 HGI complexes from Rhizoctonia solani that is used to determine the phylogenetic position of the pathogen's isolate associated with web blight on okra.New Zealand), to determine quality and to edit.Then, we aligned them using the ClustalX software (THOMPSON et al., 1997), and we finally compared them to sequences of the rDNA's ITS region from all R. solani's anastomosis groups deposited in the GenBank (NCBI) database.We searched for similar sequences using BLASTN (nucleotide-nucleotide), version 2.2.27 from September 10, 2012 (ALTSCHUL et al., 1997).The isolate's sequence was coded MF497483 in the GenBank.Due to its similarity to sequences of the rDNA's ITS-5.8Sregion of R. solani's AG-1 anastomosis group, we conducted a phylogenetic analysis comparing the AG-1 IA, IB, IC, ID, IE and IF groups -already described in Brazil -, using R solani's AG-4 HGI as an outgroup, for the tree rooting.The sequences used are described in Table 1.The phylogenetic analysis was performed using the Geneious R9.1.8software and its tool Geneious Tree Builder, as well as the HKY evolution model and the UPGMA method to build the tree.
To obtain the consensus phylogenetic tree, data were resampled through bootstrapping with 1,000 replicates.
Considering the morphologic characteristics and the phylogenetic position of the okra isolate's rDNA ITS-5.8Sregion (Fig. 3), which features sequences identical to AG1 ID, T. cucumeris (asexual phase of R. solani AG-1 ID) was the fungus identified as the agent causing the disease.This is the first report of an association between R. solani AG-1 ID and okra.The occurrence of R. solani AG-1 ID as a pathogen associated with web blight, however, was reported in Amazonia during an association with passion fruit (Passiflora edulis f. flavicarpa Deg., Bel59 isolate), beans (Phaseolus vulgaris L., Bel60), shellflower [Alpinianutans (L.) Roscoe, Bel61], and black pepper (Piper nigrum L., Bel71) in 2010 (GAINO et al., 2010).
In the Amazon, T. cucumeris causes web blight in several hosts, such as Para rubber tree (Hevea spp.) (DESLANDES, 1944) and orange (Citrus spp.) (LOURD et al., 1984).According to CAMPOS (2006) and GAINO et al. (2010), the Para rubber tree hosts several other anastomosis groups (AGs), among them AG2-2 Hb, the most frequent one, and AG-1 1D and AG-1 IF (recently reclassified, now encompasses isolates previously classified as AG-1 IB).These isolates infect Para rubber tree and occur in other hosts (native or in crops).This wide array of hosts reflects the pathogen's potential to infect different crops.
In okra, the pathogen had been reported to cause pre-and post-emergence rots, damping off (MASSOLA; BEDENDO, 2005), and post-harvest fruit rots (HENZ et al., 1996).A strong attack of T. cucumeris happened throughout the okra plant's developmental stages, causing defoliation.It was more frequent during the rainy period (from January to May), when the conditions for infection are more favorable.This is the first report of T. cucumeris causing web blight and strong defoliation in okra in Brazil, and probably in the world.

Figure 1 .
Figure 1.Symptoms of web blight (Thanatephorus cucumeris) on the adaxial surface of okra leaves.

Figure 2 .
Figure 2. Abaxial surface of an okra leaf showing web blight symptoms and signs of the Thanatephorus cucumeris pathogen that is characterized by the growth of the pathogen's mycelium.

Figure 3 .
Figure 3. Phylogenetic analysis (UPGMA distance) of the rDNA's ITS-5.8Sregion from Rhizoctonia solani isolates of the AG-1 anastomosis group (AG-1 IA, IB, IC, ID, IE and IF) to identify the isolate associated with web blight on okra (MF497483 sequence, in bold) at Manaus, Brazil.Sequences obtained from R. solani AG-4 HGI isolates were used as outgroup.Consensus tree was obtained by bootstrap for 1,000 replicates.The values in branches indicate the bootstrap support in percentage.