Rhizoctonia solani AG4 causes lentil damping-off in Brazil
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Rhizoctonia solani is recorded here, for the first time, as a lentil pathogen in Brazil. It was isolated from lentil (Lens culinaris) affected by root rot and damping-off. The identity of the fungus was elucidated through a combination of morphological and molecular approaches. Koch’s postulates were fulfilled and pathogenicity was confirmed.
KeywordsFungal pathogen Occurrence Pulse disease Root rot
Homogeneous pure cultures were obtained either by transferring clean hyphal fragments to potato dextrose-agar (PDA) plates with a sterile fine pointed needle or by plating selected surface chlorox-disinfected diseased fragments of roots or stems onto PDA plates. Pure culture colonies were obtained and grown on PDA at 25 °C under a 12 h light regime for 7 days for observation of culture morphology. One representative isolate was deposited in the local culture collection (Accession No COAD 2089).
Slides were mounted in lactoglycerol. The fungus had the following morphology: Hyphae cylindrical, becoming monilioid with age, 2.5 to 8 μm diam, often branching at a 90 ° angle and presenting a septum next to the branching point of branches, walls hyaline becoming pale brown in older parts of colonies (Fig. 1c); Sclerotia irregularly shaped, grayish sepia. Mycelium was stained with SYBR® Green (Sigma-Aldrich) and observed with a Olympus BX 50 fluorescence microscope and cells were found to be multinucleate (Fig. 1d). This morphology was found to be equivalent to that described by Parmeter and Whitney (1970) for Rhizoctonia solani.
To confirm the morphology based identification, genomic DNA of COAD 2089 was extracted using the Wizard Genomic DNA Purification Kit (Promega) by following the manufacturer’s instructions. The internal transcribed spacer (ITS) region of ribosomal DNA was PCR amplified with the universal primers ITS4 and ITS5 (White et al. 1990) and sequenced by Macrogen (Korea). The resulting sequence was deposited in GenBank (accession no. MH259594). This sequence had 99% identity with an isolate of R. solani AG4-HG-I (Accession No. HG934415).
A pathogenicity test was performed using sixteen 30-day-old healthy lentil plants grown in 2-l pots. These were inoculated by placing one 0.5 cm diam disc taken from 7-day-old cultures grown on PDA next to the base of each of the stems. Eight healthy potted plants belonging to the same group were treated similarly to the inoculated plants but only non-colonized PDA disks were placed next to their stem base. These plants were then covered with a moistened plastic bag for 24 h and maintained in a greenhouse at ca. 28o C and irrigated twice a day. Damping-off symptoms appeared four days after inoculation on all inoculated plants. This was followed, two days later, by abundant formation of mycelium. Controls remained healthy. Typical R. solani colonies were obtained upon isolations from diseased test-plants.
Although R. solani is known to be a broad spectrum pathogen attacking many crops in Brazil, the sole previous record of R. solani on lentils in Brazil deals with its detection on seeds (Madeira et al. 1988) and does not include any information on its pathogenic status. Although Taylor et al. (2007) listed collar rot, caused by R. solani as a minor disease of the crop, Giordano et al. (1988) considered R. solani as causing the worst disease problems for lentil plantations in mid-western Brazil. Nevertheless, this publication did not provide complete details on the taxonomy of the fungus or its pathogenicity. To our knowledge this is the first time R. solani has been fully reported and characterized as a lentil pathogen in Brazil.
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