First report of Candidatus Phytoplasma australasia (16SrII- subgroup D) associated with virescence of Chia (Salvia hispanica L.) from India.

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 - 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

First Report of Powdery Mildew Caused by Erysiphe diffusa on Cluster Bean in India.

Cluster bean (Cyamopsis tetragonoloba (L.) Taub.) is an important vegetable crop cultivated widely in India. During a field survey in November 2021, about 60% of plants exhibited characteristic powdery mildew disease symptoms and signs in a 15 ha field in Northern Karnataka (Raichur), India. Initially, the symptoms and signs appeared as tan lesions, which later became small, circular and chlorotic. The abaxial surface turned yellow and was covered with white mycelial growth. As the disease progressed, white mycelia grew on the adaxial leaf surface, stems and pods as well. In severe infections, drying and premature defoliation of infected leaves were observed. Infected leaf samples with mycelia were collected (n=8) and the fungus was subjected to morphological and molecular observations. Mycelia on leaves was characterized as epiphytic, amphigenous, producing dense, white patches on the upper and lower leaf surfaces, stem and young pods. Hyphae were hyaline, thin-walled, 1.8 to 4.2 µm wide with erect conidiophores consisting of a cylindrical foot-cell, straight flexuous at the base and measured 20 to 36 × 6 to 9 µm (n=30), followed by 1 to 2 shorter cells. Ellipsoid conidia were produced singly and measured 28 to 42 × 12 to 20 µm (n=30) without fibrosin bodies. Chasmothecia were not observed. A reference specimen was deposited at the Institution of Excellence, University of Mysore Herbarium (UOM-IOE 2022_1). The morphology and other characteristics of conidia were consistent with an Erysiphe species (Braun and Cook 2012). Genomic DNA was isolated from a conidial suspension harvested from the powdery mildew affected cluster bean samples. The ITS region was amplified from three samples using powdery mildew-specific primer pair PN23/PN34 and sequenced directly (Chen et al. 2008). nBLAST analysis revealed that the ITS sequence shared 100% similarity with the reference sequence (E. diffusa vouchers HMJAU02177 - KM260363, BRIP 71013 - MW009058) of Erysiphe diffusa (Cooke & Peck) U. Braun & S. Takam. In addition to 100% match to voucher specimens of E. diffusa, there were no vouchers from other species that also had 100% match. The representative sequences were deposited in GenBank with accession numbers OM669776 - OM669778. Koch's postulates were conducted on healthy cluster bean plants grown under greenhouse conditions. Conidia were harvested from infected leaves, suspended in water and sprayed on 40 to 50-day-old cluster bean plants (28 ± 2°C and >70% relative humidity). The development of powdery mildew symptoms was recorded on 22 plants after 10-14 days of post inoculation. Control plants inoculated with sterile water remained healthy without powdery mildew symptoms. Microscopic observation of spores from inoculated plants confirmed the pathogen as E. diffusa. The genus Erysiphe is known to infect many crop plants. E. diffusa has been reported to infect Vigna radiata, Glycine max and Phaseolus mungo in Australia (Kelly et al. 2021). No reports are available at USDA's host-fungus database for cluster bean and E. diffusa (Farr and Rossman 2022). To the best of our knowledge, this is the first report of E. diffusa associated with powdery mildew of cluster bean in India. Further comprehensive investigations will shed a light on the economic impact of powdery mildew disease on the cluster bean in India.

A new host record for Candidatus Phytoplasma cynodontis (16Sr XIV-A) associated with phyllody and fasciation of linseed (Linum usitatissimum) from India.

Linseed commonly called as flaxseed (Linum usitatissimum Linn.) is an important oilseed crop cultivated widely in Northern parts of Karnataka. During, 2019 (January-February), a characteristic disease was noticed with symptoms that resembled phytoplasma or like disease symptoms. The incidence was ranged from 6·5 to 16·5% in the experimental station of Raichur Agricultural University. The typical symptoms observed were virescence of floral parts, fasciation of the inflorescence axis, phyllody, stunted and flattened stem with reduced leaves. Symptomatic and healthy samples were collected and processed for molecular detection of phytoplasma. Total DNA was isolated from four infected plants and two healthy plants. The 16S rDNA region was amplified using P1/P7 followed by R16F2n/R16R2 primer pair which showed the amplification of expected amplicon size from all four infected samples. Furthermore, the SecA gene was amplified using SecA1/SecA3 primers. The PCR amplified products were subjected for direct sequencing from both directions and the consensus sequences were obtained and nBLAST search analysis revealed that the 16Sr RNA and SecA sequences were sharing maximum similarity (100%) with the reference sequence of Ca. P. cynodontis. The sequences were analysed phylogenetically by constructing a Phylogram independently by NJ method along with reference sequence of 16S rRNA region and SecA region retrieved from GenBank database showed that the phytoplasma sequence from linseed phyllody of the present study placed in a distinct clade along with reference sequence of "Ca. P. cynodontis" thus confirming the identity phylogenetically. Furthermore, iPhyClassifier and virtual RFLP proved that the phytoplasma belonged to 16SrXIV (subgroup A) phytoplasma. Previously linseed is known to be associated with 16SrII-D phytoplasma but the association of the 16SrXIV-A group of phytoplasma is not reported so far. Therefore, this is the new host record for Ca. P. cynodontis (16SrXIV-A) phytoplasma associated with linseed stem fasciation, phyllody from India.

A Study of Hair Follicular Transplantation as a Treatment Option for Vitiligo.

BACKGROUND: Repigmentation of vitiligo is closely related to hair follicles. Hence, replenishing melanocytes in vitiliginous patches utilizing undifferentiated stem cells of the hair follicles using follicular unit transplantation (FUT) is a possible treatment option. OBJECTIVES OF THE STUDY: To study the efficacy of FUT in cases of segmental/stabilized vitiligo as a treatment option for leukotrichia. MATERIALS AND METHODS: Fifty patients with 63 lesions of stable vitiligo over nonglabrous areas were treated with follicular unit grafts. Reduction in the size of vitiligo patches as well as improvement in the associated leukotrichia were evaluated using subjective and objective assessments. RESULTS: Of the 63 patches, good to excellent response was seen in 39 (61.9%), fair in 16 (25.4%), and poor in eight (12.7%) lesions. No repigmentation was seen in two (4.8%) lesions. The mean improvement seen was 61.17%. Excellent color match was observed in 44 lesions (69.8%). Repigmentation of the depigmented hairs occurred in 11 out of 46 patients with associated leukotrichia. CONCLUSION: FUT is a safe and effective method for treating localized and segmental vitiligo, especially on hairy parts of the skin. Though labor intensive, it was found to be associated with a quick patient recovery time, very low morbidity, and good color match.