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.

Fungal Planet description sheets: 1436-1477.

Novel species of fungi described in this study include those from various countries as follows: Argentina, Colletotrichum araujiae on leaves, stems and fruits of Araujia hortorum. Australia, Agaricus pateritonsus on soil, Curvularia fraserae on dying leaf of Bothriochloa insculpta, Curvularia millisiae from yellowing leaf tips of Cyperus aromaticus, Marasmius brunneolorobustus on well-rotted wood, Nigrospora cooperae from necrotic leaf of Heteropogon contortus, Penicillium tealii from the body of a dead spider, Pseudocercospora robertsiorum from leaf spots of Senna tora, Talaromyces atkinsoniae from gills of Marasmius crinis-equi and Zasmidium pearceae from leaf spots of Smilaxglyciphylla. Brazil, Preussia bezerrensis from air. Chile, Paraconiothyrium kelleni from the rhizosphere of Fragaria chiloensis subsp. chiloensis f. chiloensis. Finland, Inocybe udicola on soil in mixed forest with Betula pendula, Populus tremula, Picea abies and Alnus incana. France, Myrmecridium normannianum on dead culm of unidentified Poaceae. Germany, Vexillomyces fraxinicola from symptomless stem wood of Fraxinus excelsior. India, Diaporthe limoniae on infected fruit of Limonia acidissima, Didymella naikii on leaves of Cajanus cajan, and Fulvifomes mangroviensis on basal trunk of Aegiceras corniculatum. Indonesia, Penicillium ezekielii from Zea mays kernels. Namibia, Neocamarosporium calicoremae and Neocladosporium calicoremae on stems of Calicorema capitata, and Pleiochaeta adenolobi on symptomatic leaves of Adenolobus pechuelii. Netherlands, Chalara pteridii on stems of Pteridium aquilinum, Neomackenziella juncicola (incl. Neomackenziella gen. nov.) and Sporidesmiella junci from dead culms of Juncus effusus. Pakistan, Inocybe longistipitata on soil in a Quercus forest. Poland, Phytophthora viadrina from rhizosphere soil of Quercus robur, and Septoria krystynae on leaf spots of Viscum album. Portugal (Azores), Acrogenospora stellata on dead wood or bark. South Africa, Phyllactinia greyiae on leaves of Greyia sutherlandii and Punctelia anae on bark of Vachellia karroo. Spain, Anteaglonium lusitanicum on decaying wood of Prunus lusitanica subsp. lusitanica, Hawksworthiomyces riparius from fluvial sediments, Lophiostoma carabassense endophytic in roots of Limbarda crithmoides, and Tuber mohedanoi from calcareus soils. Spain (Canary Islands), Mycena laurisilvae on stumps and woody debris. Sweden, Elaphomyces geminus from soil under Quercus robur. Thailand, Lactifluus chiangraiensis on soil under Pinus merkusii, Lactifluus nakhonphanomensis and Xerocomus sisongkhramensis on soil under Dipterocarpus trees. Ukraine, Valsonectria robiniae on dead twigs of Robinia hispida. USA, Spiralomyces americanus (incl. Spiralomyces gen. nov.) from office air. Morphological and culture characteristics are supported by DNA barcodes. Citation: Tan YP, Bishop-Hurley SL, Shivas RG, et al. 2022. Fungal Planet description sheets: 1436-1477. Persoonia 49: 261-350. https://doi.org/10.3767/persoonia.2022.49.08.

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.

Doxorubicin-DNA adduct entrenched and motif tethered artificial virus encased in pH-responsive polypeptide complex for targeted cancer therapy.

Doxorubicin is a broad spectrum anticancer antibiotic that possesses toxic effects such as cardiomyopathy, that even lead to congestive heart failure. Thus, the development of a new bio-inspired system is required, that retain the advantageous effect of doxorubicin while retarding the side effects. Hence, a system was developed that we describe 'doxorubicin-DNA adduct entrenched artificial virus encased in polypeptide complex'. The drug-DNA adduct (DDA) was prepared by a formaldehyde mediated reaction. A simple chloroform extraction method for the separation of DDA was developed. DDA was employed to self-assemble the folate tethered bovine serum albumin to form the protein coat in the proposed artificial virus. The folate tethered albumin provides an artificial virus concept, with tumor tissue targeting due to the presence of folate. The whole system was then encased in a pH-responsive polypeptide complex that dissolves in acidic pH, but not in basic pH. DDA was evaluated by UV-Vis spectrophotometry, spectrofluorimetry and high-performance liquid chromatography (HPLC). A promising drug release at physiological condition was observed from DDA. The developed system was evaluated by a developed and validated artificial cell apparatus that mimic the features of a cancer cell. The drug delivery system displayed a considerable amount of drug release within 24 h. Moreover, the developed artificial virus system reduced angiogenesis caused by tumor cells in chick chorioallantoic membrane. Histopathology of treated chicken heart slices demonstrated that the developed artificial virus system reduces the tissue deformation and apoptosis in heart tissue slices, thus providing a new approach to prevent Dox-induced cardiomyopathy.

Artificial Virus as Trump-card to Resolve Exigencies in Targeted Gene Delivery.

Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized to successfully deliver the genetic material. However, the use of virus mediated gene delivery is considered divisive, because the potentially replicable genomes recombine or integrate with the cell DNA resulting in immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective non-viral gene delivery vehicles arises. Non-viral vectors, protein only particles and virus like particles (VLP) can be constructed which contain all the necessary functional moieties. These resemble viruses and are called artificial or synthetic virus. The artificial virus eliminates the disadvantages of viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because incorporation of requisite agents such as cell ligands, membrane active peptides, etc. into proteins is possible. The protein- DNA complexes resemble bacterial inclusion bodies. Nucleic acids influence conformation of protein units which subsequently result in cell uptake and finally to the cell nucleus. Such tunable systems mimic the activities of infected viruses and are used for the safe and effective delivery of drugs and genetic material in gene therapy. The versatility, stability and biocompatible nature of artificial virus along with high transfection efficacy have made it favorite for gene delivery purposes, in addition to being useful for various biomedical and drug delivery applications.

Concept of Health Care Counseling for Pediatricians.

Health care counseling (HCC) is a relatively new concept that amalgamates human biology, human psychology and medical sociology principles, and applies the same in real-time clinical situations. In India, there is a real paucity of trained mental health personnel, and hence counseling services are restricted to few departments. HCC is especially important for the child population, as the pediatricians need to partner the parenting responsibilities in different illness care settings covering the period from newborn to adolescence. This paper proposes steps for further development of the concept, expertise and systematic training program for health personnel, as an activity of Centre for Health Care Counseling Studies under Kerala University of Health Sciences. Once the process is documented, we hope that the same would be made available to other states in India.

Deltamethrin resistance in south Indian isolates of Rhipicephalus (Boophilus) microplus.

The present communication deals with the detection and characterization of deltamethrin resistance in tick populations using biological (larval packet test), biochemical (esterase enzyme assay) and molecular assays. Ticks were collected from cattle farms of Korutla, Telangana (KOR), Mehboob Nagar, Telangana (MBN), Nagpur, Maharashtra (NAG), Parbani, Maharashtra (PBN), Madhavaram, Tamil Nadu (MAD), Cuddalore, Tamil Nadu (CUD), Sakhleshpur, Karnataka (SAK) and Buvenduvella, Karnataka (BUV). Out of eight field isolates, seven were identified as Rhipicephalus (Boophilus) microplus while one isolate (CUD) was identified as R. (B.) annulatus. The LC50 values and resistance factors (RF) of field isolates were assessed by larval packet test (LPT). RF values of two isolates viz., Korutla and Parbhani (KOR, PAR) were close to that of reference susceptible isolate. R. (B.) microplus isolate from Nagpur (NAG) and Sakleshpur (SAK) revealed slightly higher RF values (6.42 and 4.51). They revealed slightly elevated esterase enzyme activity too. Other isolates did not reveal higher values for RF or esterase activity. Previously identified mutations conferring synthetic pyrethroid resistance in R. (B.) microplus populations were analysed by sequencing the mutation flanking regions of the carboxyl esterase and the sodium channel genes (domain III S6 and domain II S4-5 linker region). However, these point mutations were not detected in the field isolates. The results of the present study revealed that low levels of synthetic pyrethroid resistance had developed in field populations of ticks of southern India.

Histoarchitecture of ovary of Haemaphysalis bispinosa during engorgement period.

The ovary of Haemaphysalis bispinosa was of panoistic type with asynchronous development of oocytes. The wall of the ovary was composed of a layer of epithelial cells to which the oocytes were attached by means of pedicel cells with elongated nucleus. The oocytes were classified into stages I to V based on morphologic characteristics like size and shape, presence / absence of germ vesicle, cytoplasmic appearance, presence or absence of yolk granules and presence of chorion. Day wise changes were in the form of occurrence of oogonia from partially fed upto day zero of engorgement, presence of all stages of oocytes on day one and two after engorgement and onset of degenerative changes in oocytes from day three onwards. Degeneration was complete on day eight with the appearance of polymorphism, vacuolation, cytoplasmic blebbing and autophagic activity in oocytes.

Histoarchitecture of ovary of Haemaphysalis bispinosa during engorgement period

The ovary of Haemaphysalis bispinosa was of panoistic type with asynchronous development of oocytes. The wall of the ovary was composed of a layer of epithelial cells to which the oocytes were attached by means of pedicel cells with elongated nucleus. The oocytes were classified into stages I to V based on morphologic characteristics like size and shape, presence / absence of germ vesicle, cytoplasmic appearance, presence or absence of yolk granules and presence of chorion. Day wise changes were in the form of occurrence of oogonia from partially fed upto day zero of engorgement, presence of all stages of oocytes on day one and two after engorgement and onset of degenerative changes in oocytes from day three onwards. Degeneration was complete on day eight with the appearance of polymorphism, vacuolation, cytoplasmic blebbing and autophagic activity in oocytes.

Low level deltamethrin resistance in ticks from cattle of Kerala, a south Indian state.

The deltamethrin resistance status in Rhipicephalus (Boophilus) annulatus and R. (B.) microplus ticks collected from cattle of five organized farms of Kerala, south India was evaluated. Resistance was characterized using biological (larval packet test), biochemical (esterase enzyme activity assay) and molecular tools (PCR amplification and sequencing of deltamethrin resistance-associated genes). Characterization of field isolates revealed level I resistance in ticks collected from four out of five farms. Elevated level of α/ß esterase activity was not recorded in isolates showing level I resistance. Previously reported point mutations in the carboxyl esterase (G1120A) and sodium channel (T2134A and C190A) genes were not observed in any of the field isolates. The present study showed a low level (level I) resistance is developed in the most economically important ticks infesting cattle of this state and it cautions the development of large scale resistance in future.

Acaricidal activity of alkaloid fractions of Leucas indica Spreng against Rhipicephalus (Boophilus) annulatus tick.

The acaricidal activity from alkaloid and non-alkaloid fractions of Leucas indica were studied against Rhipicephalus (Boophilus) annulatus tick using adult immersion test under laboratory conditions. For this purpose, the engorged female R.(B.) annulatus tick were exposed to two fold serial dilutions of alkaloid extract (50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6 mg/ml and 3 mg/ml) using 'dipping method' in vitro. The efficacy was assessed by measuring the percentage of adult mortality, inhibition of fecundity and hatching rate. The alkaloid fraction of the extract produced concentration dependent delayed adult tick mortality. The extract at a concentration of 50 mg/ml demonstrated 66.67 per cent mortality and 55.16 per cent inhibition of fecundity. Nicotine was identified as one of the compounds of alkaloid fraction. However, it did not reveal any acaricidal activity when tested in vitro at concentrations ranging from 62.5-1000 µg/mL. Hence, the acaricidal action of L. indica is not due to nicotine. Non alkaloid fraction also did not reveal any acaricidal effects against R. (B.) annulatus tick.

Acaricidal effect of Cassia fistula Linn. leaf ethanolic extract against Rhipicephlaus (Boophilus) annulatus.

The present study evaluates the acaricidal properties of crude ethanolic extract of Cassia fistula leaves for controlling Rhipicephalus (Boophilus) annulatus based on adult immersion test (AIT). The percentage of adult mortality, inhibition of fecundity and hatching of ova laid were studied at different concentrations of the extract ranging from 50 to 100 mg / ml. The results were compared using one-way ANOVA. The extract produced complete inhibition of hatching of eggs at concentrations above 80 mg / ml of the extract. Mortality of adult engorged female ticks and inhibition of fecundity were concentration dependent. The LC50 value of extract against R. (B.) annulatus was 97.1 mg / ml.

Phylogenetic analysis of bovine Theileria spp. isolated in south India.

The objective of the present study is to determine the phylogenetic position of the Theileria organisms in blood of cattle of southern India using molecular tools. Theileria annulata (Namakkal isolate, Tamil Nadu) and three Theileria field isolates (free of T. annulata) from Wayanad, Kerala (Wayanad 1, 2, 3) were used. The small subunit ribosomal RNA (SSU rRNA) and major piroplasm surface protein (MPSP) gene products were cloned, sequenced and the phylogenetic tree constructed. SSU rRNA gene of Wayanad 1 isolate (JQ706077) revealed maximum identity with Theileria velifera or Theileria cervi. The phylogenetic tree constructed based on SSU rRNA genes revealed that Wayanad 1 isolate belonged to a new type which share common ancestor with all the other theilerial species while Wayanad 2 and 3 isolates (JX294459, JX294460) were close to types A and C respectively. Based on MPSP gene sequences, Wayanad 2 and 3 (JQ706078, JX648208) isolates belonged to Type 1 and 3 (Chitose) respectively. When, the previously reported MPSP type 7 is also considered from the same study area, Theileria orientalis types 1, 3 and 7 are observed in south India. SSU rRNA sequence of South Indian T. annulata (JX294461) showed a maximum identity with Asian isolates while the Tams1 merozoite surface antigen (MSA) gene (JX648210) showed maximum identity with north Indian isolate.

An integrated approach for the reduction of aflatoxin contamination in chilli (Capsicum annuum L.).

An integrated approach for management of aflatoxin contamination in chilli was undertaken by evaluating the fungicides, bioagents and plant extracts against Aspergillus flavus under both in vitro and field condition. Maximum inhibition of radial growth (91.1%) was observed with 0.3% mancozeb followed by captan (85.2%). Carbendazim (73%) was effective and superior over other systemic fungicides. A complete inhibition (100%) of A. flavus was observed in neem seed kernel extract (NSKE), nimbicidin and pongamia oil at 5%. An indigenous Pseudomonas fluorescens bioagent isolate inhibited (74.9%) the growth of A. flavus over Trichoderma harzianum (70.4%). The superior performing fungicides, plant extracts and bioagents identified under in vitro were used for challenge inoculation on chilli fruits and so also for field evaluation. The captan treated fruits recorded the least infection of A. flavus (1.6%) followed by P. fluorescens (2.0%), NSKE (2.2%) and nimbicidin treated fruits (7.8%) as against control (38.3%). As regards to field evaluation, the least incidence was recorded in NSKE sprayed chilli plot (1.6%) and was on par with captan (2.2%), P. fluorescens (2.4%) and T. harzianum (2.6%) compared to control (7.4%). Hence, a pre-harvest spray of NSKE (5%) or mancozeb (0.3%) or P. fluorescens (1 × 10(8) cfu/ml) 10 days before harvest of chilli is recommended for field level management of aflatoxin.

First record in South Asia of deer throat bot fly larvae Pharyngomyia picta (Meigen, 1824) (Diptera: Oesteridae) from Sambar deer (Rusa unicolor), a new host record.

The Bot fly larvae, identified to be the third instars of the deer throat bot fly Pharyngomyia picta were recovered from the lumen of trachea and secondary bronchi during the necropsy of a female sambar deer (Rusa unicolor) in Kerala, India. This forms the first report of P. picta from India and the whole of South Asia. Sambar deer is a new host record for the larvae of this fly. Morphological description of the third stage larvae with supporting figures are presented.