A. Saroj

First report of Tagetes erecta damping off caused by Ceratobasidium sp. from India.

The Mexican marigold (Tagetes erecta) is cultivated commercially in India for medicinal, ceremonial, and decorative purposes. It is native to Mexico and the United States. The natural phytochemical thiophene extracted from T. erecta has been shown to have antibacterial activity. It is also grown to extract lutein, a common yellow/orange food color. During winter of 2011, approximately 15% marigold seedlings exhibited damping off symptoms at CSIR-CIMAP, Lucknow, India, and its adjoining areas. Infected seedlings initially produced water-soaked lesions on the stem at the soil level that later turned pinkish with a brownish halo in the center. The infected seedlings were cut into small pieces, surface disinfected with 1% sodium hypochlorite, rinsed thrice with sterile distilled water, and placed onto potato dextrose agar (PDA) plates. The plates were incubated at 25°C for 3 days. The isolation yielded whitish fungal growth that later turned tan brown. The mycelium was binucleate, septate, sub branching at right angles, with distinct constriction at the origin of branching. Bisbenzamide (Hoechst 33258; Chemical Abstracts no. 23491-45-4) was used as fluorescent dye for the staining of nuclei. Based on cultural as well as morphological characteristic features, the fungus was identified as Ceratobasidium sp. (1,2). The molecular identification was on the basis of internal transcribed spacer (ITS) region sequence. Amplification of the ITS of rDNA using primers ITS1/ITS4 yielded a ~700 bp band and sequenced data were deposited in the NCBI GenBank (KC193238). The ITS region (700 bp) was 100% identical to Ceratobasidium sp. AG-F strain BAGF101 isolated from Musa spp. in Georgia, United States (GenBank Accession No. HQ168370). The pathogenicity of the fungus was tested under glasshouse conditions. The inoculum of the fungus was prepared on sterile maize seeds in Erlenmeyer flasks by inoculating seeds with three disks (1 mm) of 5-day-old culture, and kept at 25 ± 2 °C for 14 days in the dark. The healthy, 5 to 7 day old seedlings were inoculated with five artificially infested maize seeds per pot. The uninoculated seedlings served as control. Both inoculated and uninoculated seedlings were kept at 28 ± 2°C in a humidity (95%) chamber for 3 days and thereafter placed in the glasshouse at 28 ± 2°C for development of disease symptoms. Initial symptoms developed as water-soaked lesions on the infected seedlings in 2 to 3 days, while typical disease symptoms appeared after 4 to 5 days of inoculation. Uninoculated seedlings were free from infection. The fungus was reisolated from the artificially infected seedlings on PDA and its identification as Ceratobasidium sp. was confirmed by morphological and molecular characteristics. Recently, Ceratobasidium sp. was reported as causal organism of root rot on Atractylodes macrocephala and banana (3,4). To the best of our knowledge, marigold damping off disease caused by Ceratobasidium sp. has not been reported so far on T. erecta. Hence, it is the first report from India. During fungal disease management for marigold, association of Ceratobasidium sp. should not be ignored for better crop protection. References: (1) R. T. Moore. Mycotaxon 29:91, 1987. (2) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN. 1991. (3) J. Yin et al. Plant Dis. 95:490, 2011. (4) J. M. You et al. Plant Dis.97:139, 2013.

New report of a sweet basil leaf blight caused by Cochliobolus lunatus in India.

Sweet basil (Ocimum basilicum), a member of the Lamiaceae, is used as an ornamental as well as a culinary herb. It is a rich source of the phenolic compound methyl chavicol and is used as a traditional medicinal plant in India, where the crop is grown on ~2,500 ha annually (4). The species is native to India, where it has been cultivated for >5,000 years. During the rainy season, August of 2013, a severe leaf blight was observed on 30- to 45-day-old sweet basil plants in experimental fields (approximately 5 ha) at the CSIR-CIMAP and adjoining areas in Lucknow. Initial symptoms comprised small, irregular, necrotic lesions that coalesced into a leaf blight. Infected parts of the leaves turned black during wet and humid conditions. The incidence of symptoms ranged from 20 to 30%. Infected leaf samples were cut into small pieces and surface-sterilized with 1% sodium hypochlorite for 1 min, followed by two rinses in sterilized, distilled water. The leaf pieces were then blotted dry with sterilized filter paper, placed onto potato dextrose agar (PDA), and incubated at 28°C for 3 to 5 days. Blackish-brown fungal colonies developed. Microscopic examination revealed the presence of brown conidiophores that were cylindrical, septate, unbranched, and straight or geniculate near the apex. Conidia were three-septate, mostly curved at the third cell from the base, which was usually larger and darker than the other cells; intermediate cells were brown or dark brown; terminal cells were subhyaline or pale brown and 16 to 23.5 × 8.5 to 11.5 μm (the average size of 100 conidia was 19.9 × 10.18 μm). On the basis of these characteristics, the fungus was identified as Cochliobolus lunatus (anamorph Curvularia lunata (Wakk.) Boedijin) (1,2). The identification was confirmed by sequencing the internal spacer (ITS) region of ribosomal DNA (rDNA). Genomic DNA was extracted from five fungal isolates, using the 5 Prime Archive Pure DNA Cell/Tissue kit, and subjected to a polymerase chain reaction (PCR) assay with the universal primers ITS1 and ITS4 (5). The amplified product was cloned and sequenced. An NCBI-BLASTn search showed greatest homology (98% similarity) with the ITS sequence of C. lunatus (GenBank Accession No. DQ836800). The sequence was deposited in Genbank (KM272001). A pathogenicity test was carried out using 10, 30-day-old sweet basil (cv. CIM Soumya) plants in pots, by spraying a spore suspension (105 spores/ml) onto the leaves of each plant. Five plants treated similarly with sterilized, distilled water served as a control treatment. The plants were kept at 27 ± 2°C and 85 ± 3% RH for 8 to 10 days. Small, irregular, necrotic lesions appeared after 4 days on all inoculated leaves, while leaves of control plants remained asymptomatic. Fungi re-isolated from inoculated leaves resembled C. lunatus on the basis of microscopic and sequence data, fulfilling Kochs postulates. The fungus was not re-isolated from the control plants. C. guatemalensis has been reported to cause a leaf spot on sweet basil in Korea (3). To our knowledge, this is the first report of a sweet basil leaf blight caused by C. lunatus in India. C. lunatus has the potential to reduce the yield of sweet basil. References: (1) L. M. Liu et al. Plant Dis. 98:686, 2014. (2) D. S. Manamgoda et al. Fungal Divers. 56:131, 2012. (3) J. H. Park et al. Plant Dis. 96:580, 2012. (4) H. A. A. Taie et al. Not. Bot. Hort. Agrobot. Cluj. Napoca 38:119, 2010. (5) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

New Report of Black Leaf Spot Mold (Pseudocercospora fuligena) on Withania somnifera from India

Withania somnifera (family solanaceae) commonly known as ashwagandha and Indian ginseng, originated in India is one of the most powerful medicinal plants for more than 3,000 years (1). It is commercially cultivated for its roots, a natural rich source of glycowithanolides, tannins, potassium nitrate, etc., which are an anti-inflammatory, anti-tumor, anti-oxidant, anti-ulcer, and regulator of the nervous system and sleep (2). During the monsoon of July 2011, black spots on the leaves of infected plants were observed in the ashwagandha growing Lucknow, Raibareilly, and adjoining areas of Uttar Pradesh province with 10 to 20% disease incidence. Early stage of disease were characterized by the presence of light chlorotic spots on both sides of old leaves that later turned into dark black spots resulting in early defoliation. About 27 samples were collected from different locations of the fields for isolation of the causal organism and microscopic studies. Infected leaves were cut into small pieces, surface sterilized with 1% sodium hypochlorite for 1 min, rinsed thrice with sterilized distilled water, and placed onto potato dextrose agar (PDA) plates. After 21 days of dark incubation at 25°C, 8- to 10-mm grayish-brown colonies were observed. Microscopic studies at early and mature stages of infection showed production of conidia in conidiophores. Conidiophores were mostly 5 to 9, few dense pale brown, simple unbranched, septate, geniculate and 14 to 55 × 3 to 5.5 μm. Conidia were subhyline, obclavate to cylindrical, some were straight to slightly curved, multiseptate, base long obconic to long obconically truncate, and 12 to 85 × 3.5 to 5 μm. On the basis of cultural and morphological studies, the pathogen was identified as Pseudocercospora fuligena (3). The pathogen identity was further confirmed at molecular level using universal primers ITS1/ITS4 through PCR (4). An amplification of the expected size (~550 bp) was generated, eluted from agarose gel by QIAquick gel extraction kit (Qiagen), cloned into pGEM-T Easy vector (Promega), sequenced, and deposited in GenBank (Accession No. KF881898). NCBI BLASTn showed 99% identity with P. fuligena (GU214675) strain CPC 12296, isolated from Lycopersicon sp. Pathogenicity test was carried out on 10 plants of W. somnifera cv. Poshita through two approaches, one using mycelia from culture and another using spore suspension from naturally infected leaves. In the first approach, fungal mycelia were applied onto the healthy ashwagandha leaves, whereas in the second approach, infected leaves were washed with distilled water and spore suspension of 106 spores/ml was sprayed on healthy plants. Plants sprayed with sterilized distilled water served as controls. Inoculated plants were placed in a growth chamber at 28°C under 90% humidity for 3 days. After, pots were placed in the glasshouse at 27 ± 2°C with 70 to 80% humidity for 21 days. Initial symptoms appeared on the 7th day while typical symptoms appeared on all the inoculated plants after 12 to 17 days. Control plants remained free of infection. Re-isolation of the pathogen on PDA fulfilled Kochs postulates. Black leaf mold caused by P. fuligena has been reported on tomato (5). This is the first report of black leaf mould caused by P. fuligena on W. somnifera from India. P. fuligena has the potential to reduce yield of W. somnifera. References: (1) Anonymous. Alt. Med Rev. 9:211, 2004. (2) B. D. Basu and K. R. Kirtikar. Indian Medicinal Plants: Plates, vol. 1-4. Bishen Singh Mahendra Pal Singh, Dehradun, India, 1991. (3) T. C. Wang et al. Plant Dis. 79:661, 1995. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (5) S. Yamada. Ann. Phytopathol. Soc. Jpn. 15:13, 1951.