Diaporthe phaseolorum causing dieback disease on Melia dubia cav. in Karnataka state (India).

Melia dubia is an important tree species grown worldwide for its medicinal and timber values. It is widely used in timber and pulp industry and also as an organic pesticide, fertilisers, agro-forestry and herbal formulations. During 2019-2022, a dieback disease in plantations of M. dubia was recorded in Mysore, Mandya, Chamarajanagar, Hassan and Tumkur districts of Karnataka state (India) with disease incidence of 26.25%. The associated pathogen was isolated on PDA medium and its morpho-cultural characteristics were studied. The genomic DNA of the pathogen was isolated, and rDNA was amplified and sequenced using universal primers. Based on the microscopic, morpho-cultural, sequence data and phylogenetic analysis, the pathogen was identified as Diaporthe phaseolorum (Cooke & Ellis) Sacc. Koch's postulates were performed both in vitro and in vivo and the typical symptoms of dieback disease were recorded on post-inoculated saplings. The dieback disease is responsible for the poor growth of Melia species in the region, and hence, there is an urgent need to manage the disease in plantations using integrated management practices. This is the first report of the occurrence of D. phaseolorum on M. dubia plantations in India.

Molecular detection and characterization of Potato spindle tuber viroid (PSTVd) infecting Tomato (Solanum lycopersicum L.) in Karnataka State of India.

Potato Spindle Tuber Viroid (PSTVd) is a non-coding, infectious, small, circular RNA known to cause disease in agricultural and horticultural plants. In the present work, an investigation was conducted in the southern districts of Karnataka state to assess the possible pospiviroid infections on tomato plants that are considered natural hosts for viroids. A total of 83 tomato samples showing disease symptoms (virus or viroid-like) along with healthy ones were collected and subjected to viroid detection by conventional Reverse Transcriptase Polymer Chain Reaction (RT-PCR) using universal (Pospi1-RE/Pospi1-FW) and a specific set of primers (3H1/2H1). The study confirmed the presence of PSTVd in one of the samples of tomato collected from Banghatta village of Mandy District, with an expected amplicon of ~ 361 bp. The bioassay conducted on tomato plants (cv. Rutgers) proved the association of PSTVd, which was further confirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative sequences were deposited in the NCBI GenBank. The sequence alignment and secondary structure analysis of the isolated viroid with other reference sequences revealed the variations in the pathogenicity, central conserved region, and Terminal right domains. The variations observed between the isolated PSTVd with that of other Indian isolates support that viroid may have been transmitted among the crop plants, possibly through seed or mechanical means.

A New Rust Disease on Wild Coffee (Psychotria nervosa) Caused by Puccinia mysuruensis sp. nov.

Psychotria nervosa, commonly called "wild coffee" (Rubiaceae), is an important ethno-medicinal plant in India. In 2010, a new rust disease of P. nervosa was observed in three regions of Mysore District, Karnataka (India), with disease incidence ranging from 58 to 63%.Typical symptoms of the rust disease on wild coffee were prominently visible during the early monsoon season (May to June), with chlorotic spots on the adaxial and black pustules (telia) on the abaxial leaf surface. Telia produced abundant teliospores, which were bicelled, pedicillate, and measured 33 to 45 by 19 to 30 µm. The germination of teliospores produced a typical metabasidium bearing four basidiospores, each containing two haploid nuclei. Spore stages of the wild coffee rust pathogen were studied using artificially inoculated healthy wild coffee plants with germinated teliospores. Only telia were observed on the inoculated plants, indicating that this rust fungus has an abbreviated microcyclic life cycle that includes only teliospores and basidiospores. Phylogenetic analysis based on internal transcribed spacer and partial large subunit (LSU) sequence data showed that the wild coffee rust pathogen is related to Macruropyxis fraxini, Puccinia bartholomaei, P. choridis, and P. sparganioidis. The herbarium sample of P. psychotriae was examined and was shown to be different with respect to telium size and teliospore dimensions (24 to 32 by 13 to 18 µm). Therefore, the rust pathogen causing wild coffee rust is a new species, P. mysuruensis sp. nov.

First Report of Phomopsis citri Associated with Dieback of Citrus lemon in India.

Lemon (Citrus lemon (L.) Burm. f.) is an important fruit crop cultivated worldwide, and is grown practically in every state in India (3). During a survey conducted in 2013, a few small trees in a lemon orchard near Mysore city (Karnataka) (12°19.629' N, 76°31.892' E) were found affected by dieback disease. Approximately 10 to 20% of trees were affected as young shoots and branches showed progressive death from the apical region downward. Different samples were collected and diagnosed via morphological methods. The fungus was consistently isolated from the infected branches when they were surface sanitized with 1.5% NaOCl and plated on potato dextrose agar (PDA). Plates were incubated at 26 ± 2°C for 7 days at 12/12 h alternating light and dark period. Fungal colonies were whitish with pale brown stripes having an uneven margin and pycnidia were fully embedded in the culture plate. No sexual state was observed. Pycnidia were globose, dark, 158 to 320 µm in diameter, and scattered throughout the mycelial growth. Both alpha and beta conidia were present within pycnidia. Alpha conidia were single celled (5.3 to 8.7 × 2.28 to 3.96 µm) (n = 50), bigittulate, hyaline, with one end blunt and other truncated. Beta conidia (24.8 to 29.49 × 0.9 to 1.4 µm) (n = 50) were single celled, filiform, with one end rounded and the other acute and curved. Based on the morphological and cultural features, the fungal pathogen was identified as Phomopsis citri H.S. Fawc. Pathogenicity test was conducted on nine healthy 2-year-old lemon plants via foliar application of a conidial suspension (3 × 106); plants were covered with polythene bags for 6 days and maintained in the greenhouse. Sterile distilled water inoculated plants (in triplicate) served as controls and were symptomless. Development of dieback symptoms was observed after 25 days post inoculation and the fungal pathogen was re-isolated from the inoculated lemon trees. The internal transcribed spacer region (ITS) of the isolated fungal genomic DNA was amplified using universal-primer pair ITS1/ITS4 and sequenced to confirm the species-level diagnosis (4). The sequence data of the 558-bp amplicon was deposited in GenBank (Accession No. KJ477016.1) and nBLAST search showed 99% homology with Diaporthe citri (teleomorph) strain 199.39 (KC343051.1). P. citri is known for its association with melanose disease of citrus in India, the United States, and abroad. P. citri also causes stem end rot of citrus, which leads to yield loss and reduction in fruit quality (1,2). Dieback disease is of serious concern for lemon growers as it affects the overall productivity level of the tree. To the best of our knowledge, this is the first report of P. citri causing dieback of lemon in India. References: (1) I. H. Fischer et al. Sci. Agric. (Piracicaba). 66:210, 2009. (2) S. N. Mondal et al. Plant Dis. 91:387, 2007. (3) S. P. Raychaudhuri. Proc. Int. Soc. Citriculture 1:461, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Pestalotiopsis Species Causing Leaf Spot of Cowpea (Vigna unguiculata) in India.

Cowpea (Vigna unguiculata (L.) Walp) is an important legume crop cultivated in arid and semi-arid regions in underdeveloped and developing countries. India is a leading cowpea producer. In addition to India, Nigeria and Niger are the predominant producers of cowpea in the world. Brazil, Haiti, Myanmar, Sri Lanka, and the United States are also significant producers of cowpea. This is a drought-tolerant annual crop that thrives in warm weather (3), and is more well-adapted to the drier regions of the tropics than any other legume. Cowpea fields (190 ha) surveyed in Mysore district (Karnataka State) from 2010 to 2012 were found affected by a new leaf spot disease. Over 60% of surveyed fields were affected by this disease, with individual fields ranging from 30 to 75% disease incidence. Individual fields experienced an estimated 10 to 15% yield loss. Initially, leaf spot symptoms appeared as small, dark, necrotic lesions that increased to a diameter of 0.5 to 1.0 cm. These spots later enlarged to form brown, circular, elliptical, and irregular spots with halo margins. Symptoms persisted throughout the cropping season. Under severe infection, defoliation occurred. Black, sessile, discoid conidiomata were observed in lesions and exuded a pink spore mass that later turned brown. The fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl2 solution, washed thrice with sterile water, blotter dried, and inoculated onto potato dextrose agar (PDA). White mycelia produced black globular acervuli with conidia on PDA after 7 days of incubation at 28 ± 2°C with a 12-h alternate light and dark period. Conidia had 5-celled (21.37 to 24.89 × 6.3 to 6.9 µm) segmentation with darker median cells and hyaline end cells. The apical cell typically had three appendages (sometimes 2 to 4) measuring 22.0 to 27.3 µm long and the basal appendage was 3.47 to 6.2 µm long. Based on these morphological features, the fungal pathogen was identified as Pestalotiopsis species. The isolated fungus was tested for pathogenecity on 30-day-old healthy cowpea plants grown under greenhouse conditions. A conidial suspension was prepared from 7-day-old culture by flooding with 2 to 4 ml of sterile distilled water. Spores were collected with a sterile micropipette and spore concentration was adjusted to 3 × 106 conidia/ml and applied as foliar spray onto 15 plants each in three replicates. Non-inoculated control plants were sprayed with sterile water. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). After 10 to 12 days post-inoculation, leaf spot symptoms appeared on inoculated plants, and the fungal pathogen was re-isolated and no such symptoms were found on control plants. The pathogen was confirmed by micro-morphological features. The ITS region of the ribosomal RNA gene was amplified using primers ITS1 and ITS4 (2). The amplified PCR product was purified and sequenced. nBLAST search comparison of sequences revealed 99% homology to Pestalotiopsis photiniae (AY682946.1). A representative sequence was deposited in GenBank (KC568288.1). Pestalotiopsis is an important pathogen on many crop plants and has been recorded on a wide variety of hosts, primarily on leaves, fruits, and in the rhizosphere. In recent times, cowpea is susceptible to a wide range of fungal pathogens causing severe yield loss at all stages of growth and development (1). Leaf spot caused by Pestalotiopsis species are becoming a major constraint for cowpea production in India. No previous reports are available on Pestalotiopsis species causing leaf spot of cowpea in India. References: (1) S. Mahadevakumar and G. R. Janardhana. New Dis. Rep. 25:17, 2012. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, page 315. Academic Press, San Diego, 1990. (3) A. A. Zohri et al. Korean Mycol. 20:252, 1992.

First Report of Leaf Spot Disease Caused by Epicoccum nigrum on Lablab purpureus in India.

Lablab purpureus (L.) Sweet (Indian bean) is an important pulse crop grown in arid and semi-arid regions of India. It is one of the most widely cultivated legume species and has multiple uses. During a September 2010 survey, we recorded a new leaf spot disease on L. purpureus in and around Mysore district (Karnataka state) with 40 to 80% disease incidence in 130 ha of field crop studied, which accounted for 20 to 35% estimated yield loss. The symptoms appeared as small necrotic spots on the upper leaf surface. The leaf spots were persistent under mild infection throughout the season with production of conidia in clusters on abaxial leaf surface. A Dueteromyceteous fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl2 solution then washed thrice, dried, inoculated on potato dextrose agar (PDA) medium, and incubated at 28 ± 2°C at 12 h alternate light and dark period for 7 days. The fungal colony with aerial mycelia interspersed with dark cushion-shaped sporodochia consists of short, compact conidiophores bearing large isodiametric, solitary, muricate, brown, globular to pear shaped conidia (29.43 to 23.92 µm). Fungal isolate was identified as Epicoccum sp. based on micro-morphological and cultural features (1). Further authenticity of the fungus was confirmed by PCR amplification of the internal transcribed spacer (ITS) region using ITS1/ITS4 universal primer. The amplified PCR product was purified, sequenced directly, and BLASTn search revealed 100% homology to Epicoccum nigrum Link. (DQ093668.1 and JX914480.1). A representative sequence of E. nigrum was deposited in GenBank (Accession No. KC568289.1). The isolated fungus was further tested for its pathogenicity on 30-day-old healthy L. purpureus plants under greenhouse conditions. A conidial suspension (106 conidia/ml) was applied as foliar spray (three replicates of 15 plants each) along with suitable controls. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). The appearance of leaf spot symptoms were observed after 25 days post inoculation. Further, the pathogen was re-isolated and confirmed by micro-morphological characteristics. E. nigrum has been reported to cause post-harvest decay of cantaloupe in Oklahoma (2). It has also been reported as an endophyte (3). Occurrence as a pathogen on lablab bean has not been previously reported. To our knowledge, this is the first report of the occurrence of E. nigrum on L. purpureus in India causing leaf spot disease. References: (1) H. L. Barnet and B. B. Hunter. Page 150 in: Illustrated Genera of Imperfect Fungi, 1972. (2) B. D. Bruten et al. Plant Dis. 77:1060, 1993. (3) L. C. Fávaro et al. PLoS One 7(6):e36826, 2012.

A sensitive and reliable RT-nested PCR assay for detection of Citrus tristeza virus from naturally infected citrus plants.

A specific and sensitive reverse transcriptase-nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance.

Antidiabetic activity of alcoholic stem extract of Nervilia plicata in streptozotocin-nicotinamide induced type 2 diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Nervilia plicata (Orchidaceae) has long been used in the antidiabetic medicinal preparations of traditional healers of Wayanad (Kerala), but recuperative potential of the plant was remained undefined. We demonstrated the regenerative potential of the plant extract on kidney affected by type 2 diabetes besides lowering blood glucose. AIM OF THE STUDY: The aim of the current study was to investigate the recuperative and regenerative potential of alcoholic stem extract of Nervilia plicata on streptozotocin-nicotinamide induced type 2 diabetic models. MATERIALS AND METHODS: Non insulin dependent diabetes mellitus (NIDDM) was induced in overnight fasted rats by intramuscular injection (IMI) of 60 mg/kg STZ and 120 mg/kg of nicotinamide after 5 min interval. Blood glucose was assessed by a glucometer, serum urea and creatinine levels were determined by diacetylmonooxime method and Jaffe reaction respectively. Kidney sections were taken and stained with Masson's tri-dye and Periodic Acid Schiff (PAS) and examined for structural changes. Also lipid peroxidation product (LPP) levels were determined as thio barbituric acid reactive substance levels (TBARS) method. RESULTS: On administration of 5 mg/kg of plant extract, blood glucose levels of the NIDDM rats showed 62.00 and 76.29% decrease in the blood glucose levels on day 0 and day 30 respectively. Damages caused to the kidney tissue were negligible or not seen. Serum urea and creatinine levels showed 61.49 and 70.96% decrease on day 30. LPP levels of kidney and pancreas showed 70.58 and 77.41% decrease respectively. CONCLUSION: These results demonstrate significant antidiabetic and regenerative potential of the Nervilia plicata, justifying the use of plant in the indigenous system of medicine. Isolation and characterisation of the compound(s) playing pivotal role in the cure would open new vistas in the therapy of type 2 diabetes.

Detection of fumonisin producing Fusarium verticillioides in paddy (Oryza sativa L. ) using polymerase chain reaction (PCR) / Detecção de Fusarium verticillioides produtor de fumonisina em arroz empregando reação de polimerase em cadeia

The study reports the occurrence of fumonisin producing Fusarium verticillioides in 90 samples of stored paddy (Oryza sativa L.)collected from different geographical regions ofKarnataka, India. Fumonisin producing F. verticillioides was identified based on micromorphological characteristics and PCR using two sets of primers. One set of primers was F. verticillioides species specific, which selectively amplified the intergenic space region of rDNA. The other set of primers was specific to fumonisin producing F. verticillioides. Eight paddy samples were positive for F. verticillioides. Eleven isolates obtained from these samples were capable of producing fumonisin.

O estudo relata a ocorrência de Fusarium verticillioides produtor de fumonisina em 90 amostras de arroz armazenado, coletado de diferentes regiões geográficas de Karnataka, Índia. F. verticillioides produtor de fumonisina foi identificado baseado em características micromorfológicas e PCR empregando dois sets de primers. Um dos sets era F. verticillioides especie-específico, que amplificava seletivamente a região do espaço intergênico do rDNA. O outro set de primers era especifico para F. verticillioides produtor de fumonisina. Oito amostras de arroz foram positivas para F. verticillioides. Onze isolados obtidos dessas amostras foram produtores de fumonisina.

Detection of fumonisin producing Fusarium verticillioides in paddy (Oryza sativa L.) using polymerase chain reaction (PCR).

The study reports the occurrence of fumonisin producing Fusarium verticillioides in 90 samples of stored paddy (Oryza sativa L.) collected from different geographical regions of Karnataka, India. Fumonisin producing F. verticillioides was identified based on micromorphological characteristics and PCR using two sets of primers. One set of primers was F. verticillioides species specific, which selectively amplified the intergenic space region of rDNA. The other set of primers was specific to fumonisin producing F. verticillioides. Eight paddy samples were positive for F. verticillioides. Eleven isolates obtained from these samples were capable of producing fumonisin.

Mycotoxin contamination of maize grains grown in Karnataka (India).

One hundred and ninety seven maize samples representing different cultivars, collected from different agroclimatic regions of Karnataka (India) were analysed for moisture content, mould incidence, ergosterol and extent of mycotoxin contamination. Moisture content determination by the hot-air oven method revealed significantly high levels of moisture content (15-18%) in 34 (17%) samples, which exceeded the permissible limit for safe storage. Ergosterol quantification by HPLC revealed the presence of ergosterol in many samples collected from rural areas of Karnataka irrespective of the moisture content. Mould enumeration based on blotter and agar plating methods revealed the association of 24 diverse species of both field and storage moulds belonging to 14 genera. Mycotoxins analyses using monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) revealed mycotoxin contamination in 69 (34.8%) samples. Maize samples with a high incidence of diverse species of moulds and alarmingly high levels of mycotoxins in many samples indicate the need for proper surveillance and monitoring exclusively for the prevention of moulds and mycotoxins in maize produce in Karnataka before it reaches the consumer.