G. R. Janardhana

A PCR-Based Assay for the Detection and Differentiation of Potential Fumonisin-Producing Fusarium verticillioides Isolated from Indian Maize Kernels

One hundred and three Fusarium isolates from maize samples collected from different districts of Karnataka state, India, were analyzed with genus-specific, species-specific, and potential fumonisin specific oligonucleotide primers. One set of genus-specific primers ITS F and ITS R based on a highly conserved ITS region of the genus Fusarium were used to differentiate Fusarium species from closely related genera. All the Fusarium species tested scored positive with the ITS pair of primers. Detection and identification of Fusarium verticillioides species was done by using a newly designed reverse primer VERT-R (5′- CGA CTC ACG GCC AGG AAA CC −3′) based on an intergenic spacer sequence (IGS) combined with an already designed forward primer VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) published previously. Out of 103 Fusarium species tested, 83 isolates of F. verticillioides scored positive for VERTF-1/ VERT-R species-specific pair of primers. Further to discriminate potential fumonisin-producing and nonproducing strains of F. verticillioides, the VERTF-1/VERTF-2 set of primers [VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) and VERTF-2 (5′-GAG GGC GCG AAA CGG ATC GG -3′)] were used. 64 isolates of F. verticillioides scored positive for VERTF-1/ VERTF-2 pair of primers. In total, three primers, one forward primer VERTF-1 and two reverse primers VERT-R and VERTF-2, were used for the confirmation of F. verticillioides up to the species level and the second pair of primers were used to confirm the potential for fumonisin production. The developed PCR assay should provide a powerful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in a population.

Study of die back disease incidence of neem in Karnataka, India and PCR based identification of the isolates

A disease survey of die back of neem was done in different agroclimatic regions of Karnataka, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die back were collected from different regions of Karnataka, India and they were further analysed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the detection of P. azadirachtae, the causative agent of die back of neem by polymerase chain reaction (PCR). Studies revealed the amplification of expected 141 bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Karnataka indicating the causal organism of die back disease on neem. Studies revealed a very high incidence of die back in most of the places of Karnataka. This is the first report on disease incidence of die back of neem. Hand held GPS was used in the study which helps in continuous monitoring of the diseased trees.

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.

Detection of Phomopsis azadirachtae from dieback affected neem twigs, seeds, embryo by polymerase chain reaction

Abstract Twigs and seeds of Azadirachta indica (Neem) infected with dieback disease, collected from different regions of Karnataka, India were analysed to determine the pathogens. Phomopsis azadirachtae, the causal agent of dieback disease of neem was found to be seed borne. Phomopsis azadirachtae was isolated on PDA and MEA from dieback-infected neem twigs, seeds and embryo. Phomopsis genus-specific primers (5.8S r-DNA) were then used for the detection of Phomopsis azadirachtae, the causative agent of dieback of neem by polymerase chain reaction (PCR). Reactions were performed on DNA isolated from twigs, seeds and embryo of dieback-affected neem trees. Studies revealed the amplification of expected 141 bp DNA in Phomopsis azadirachtae isolated from various parts of diseased trees indicating the causal organism of dieback disease on neem. Isolation and identification by conventional technique takes around 15 – 21 days, whereas the present technique is capable of detecting very low propogules within 4 – 5 days.

Assessment of the growth inhibiting effect of some plant essential oils on different Fusarium species isolated from sorghum and maize grains

The antifungal activity of essential oils from clove, cedar wood, Cymbopogon species, peppermint, Eucalyptus and neem were tested for their efficacy against nine Fusarium species, namely F. verticillioides, F. proliferatum, F. o x y spo r u m, F. a n -thophilum, F. pallidoroseum, F. sporotrichioides, F. sol ani, F. g ra m i n ea r u m and F. lateritium, isolated from maize and sorghum. The results showed that essential oils were anti-fungal at concentrations of 500–2500 ppm or higher. The oil Cymbopogon nardus (referred to as citronella oil) was inhibiting all the Fusarium species growth at 500 ppm and higher. The next most potent inhibitors, the oil from C. citratus (referred to as lemongrass oil) and peppermint oils, were fully inhibitory from a concentration of 1500 and 2000 ppm and higher, respectively. Eucalyptus and neem oils were less effective in inhibiting the growth of Fusarium species tested, irrespective of their concentration. Among the seven essential oils tested against all the Fusarium species, the citronella oil showed highest inhibitory effect (minimum inhibitory concentration, MIC) at = 1500, 2000, 1000, 500 ppm against F. verticillioides, F. oxy sp orum, F. sporotrichioides, F. lateritium mycelial growth development respectively. However clove, lemongrass oil and citronella showed highest inhibitory effect (= 1500 ppm) against F. pr o lif e ra t u m and F. pallidoro-seum respectively. Further lemongrass oil showed highest inhibitory effects at = 1500 and 500 ppm against F. a n t h o p h i -lum and F. lateritum respectively. The least MIC for F. gra -minearum was observed in peppermint oil at = 500 ppm. The results indicate that the tested toxigenic Fusarium spp. are sensitive to the essential oils, and particularly sensitive to the citronella oil. These findings clearly indicate that essential oils should find a practical application to control the growth of Fusarium species in stored maize and sorghum grains.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF SCLEROTIUM ROLFSII ASSOCIATED WITH LEAF BLIGHT DISEASE OF PSYCHOTRIA NERVOSA (WILD COFFEE)

Psychotria nervosa (Rubiaceae), commonly known as wild coffee was found affected by leaf blight disease in the forest regions of Mysore, Madikeri and Hassan districts of Karnataka state (India). Characteristic leaf blight symptoms were observed at the centre of leaf lamina of basal leaves and tiny sclerotial bodies entangled in mycelia were prominent on the adaxial leaf surface. The associated fungal pathogen was isolated and identified as Sclerotium rolfsii on the basis of morphological characters and sequence analysis of ITS region of rDNA. The sequences shared 100% similarity with reliable sequence of S. rolfsii retrieved from GenBank. The detached leaf assay produced leaf blight symptoms seven days after inoculation. S. rolfsii known to infects a wide range of host plants including forest tree species. This is the first report of S. rolfsii causing leaf blight disease of wild coffee in India.

First Report of Sclerotium rolfsii Causing Southern Blight and Leaf Spot on Common Bean (Phaseolus vulgaris) in India

Common bean (Phaseolus vulgaris L.) is an economically important crop grown worldwide. In India, it is grown for both green bean and dry pods (seeds). During 2012-2014, sudden wilt and leaf spots were observed during early growth to preflowering stage in the Mysore and Mandya districts of Karnataka State. The disease incidence was 30 to 35% in nearly 45 to 48 ha. Symptoms include wilting of foliage, tan lesions on stems at ground level, and gradually drying of the whole plant. Leaf spot symptoms on 30- to 45-day-old plants were water-soaked lesions with concentric necrotic spots (5 to 12 mm). White mycelial strands with spherical dark brown sclerotial bodies at the stem-soil interface were observed. Infected tissues were disinfected with 2% sodium hypochlorite solution for 3 min, and plated on potato dextrose agar and incubated at 28 ± 2°C. A single associated fungal pathogen was consistently isolated. It was characterized by dense, aerial, whitish cottony mycelium. Uniformly globoid sclerotia were observed after 10 to 12 days of incubation. Whitish sclerotia (1 to 3 mm) turned to dark brown at maturation. Based on etiology, and morphological and cultural characteristics, the pathogen was identified as Sclerotium rolfsii Sacc. (syn. Athelia rolfsii (Curzi) Tu & Kimbrough) (Mordue, 1974). Identity was further confirmed through PCR amplification of internally transcribed spacer (ITS) region using ITS1/ITS4 universal primer. Amplified PCR product (550 to 570 bp) was sequenced and BLASTn search comparison revealed 99% homology to Sclerotium rolfsii (GenBank Accession Nos. DQ093668 and JX914480). Representative sequence of S. rolfsii was deposited in GenBank as KP412468.1 and KP412469.1. Pathogenicity tests were conducted on 15-day-old common bean plants (cv. S-9) by soil infestation and foliar inoculation of sclerotial bodies obtained from a 12-day-old culture of S. rolfsii. The experiment was conducted on 30 plants along with control plants. Soil infestation with sclerotia resulted in wilting and crown rot symptoms with 90% disease and 100% leaf spot incidence and no such symptoms were observed on control plants. The fungal pathogen was reisolated from inoculated plants and the identity was confirmed through morphological and cultural characteristics. Southern blight and leaf spot diseases of common bean are a major constraint for its production in India. To our knowledge, this is the first report on the occurrence of S. rolfsii causing southern blight and leaf spot of common bean in India.

Screening for inhibitory activities of essential oils on the growth of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., the causal agent of leaf spot disease of Murraya koenigii L.

Seven essential oils namely clove, cedar wood, lemongrass, peppermint, eucalyptus, citronella and neem oils were tested for their inhibitory effect on spore germination, growth of germ tube and mycelial growth of Colletotrichum gloeosporioides isolated from diseased Murraya koenigii. All essential oils inhibited the germination and growth of germ tube at different concentrations. However, significant reduction in colony growth was observed with citrus, lemongrass and peppermint oils at 1000, 1500 and 2000 ppm concentrations, respectively. Citrus oil at 1360 ppm inhibited the maximum growth of the fungus followed by lemongrass oil at 1720 ppm and peppermint at 2260 ppm, respectively. The effect of essential oils on mycelial dry weight also showed antifungal activity on the growth of Colletotrichum gloeosporioides. The study revealed the possible utilisation of these essential oils for foliar spray for the management of leaf spot disease of Murraya koenigii.

First Report of the Association of the 16SrII-D Phytoplasma Subgroup With Little Leaf Disease of Crotalaria in Karnataka, India

Smooth rattle pods plants (Crotalaria pallida Aiton. [Fabaceae]) are commonly grown as fodder and green manure in tropical and subtropical regions. In India, they are commonly used as nitrogen fixer in various agro-systems and as a source of various organic residues for soil nutrition enrichment. C. pallida is also used in folk medicine to treat urinary infections and as an anti-inflammatory agent. The extracted oil from the leaves of C. pallida is known for its antimicrobial properties. In January 2016, C. pallida grown in Mysuru area, Karnataka (India) was found associated with a characteristic little leaf disease, which was suggestive of phytoplasma infection. Symptoms also included small, yellow leaves with reduced lamina, stunting, and phyllody. The disease was noticed in the wild as well as in field-grown green manure crop. Approximately 30 to 50 plants from seven fields; that is, 10 to 15 plants randomly selected in 3 to 5 rows, were visually recorded for disease symptoms. Thirty-two out of 282 plants in different fields were estimated with phyllody symptoms, resulting in disease incidence of 11%. Leaf samples from five symptom-bearing plants along with two healthy leaf samples were collected and the DNA was manually extracted using a GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA). PCR was performed to amplify the phytoplasma 16S rRNA gene using P1/P7 primers (Smart et al. 1996). The expected 1.8-kb amplicons were detected from symptomatic Crotalaria samples and none from the asymptomatic ones. The PCR products were purified, sequenced, and the consensus sequence was deposited in GenBank with accession number KX013260 (1,566 bp). It showed a 99% sequence identity with phytoplasma members of the group 16SrII ‘Candidatus Phytoplasma aurantifolia.’ The iPhyClassifier analysis (http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) revealed that the C. pallida phytoplasma (KX013260) had a coefficient of similarity of 1.0 and shared identical RFLP profiles to those of Ca. P. australasia (Y10097), which belongs to the subgroup 16SrII-D. Phytoplasmas of group 16SrII-D have been found affecting various vegetables, crops, and weeds in India, including tomato, brinjal, sesame, and parthenium. Crotalaria species are the reported hosts of several groups of phytoplasma which include 16SrVII-D (C. juncea: shoot proliferation) from Brazil (Flores et al. 2013); 16SrII-A (C. szemaoensis, C. zanzibarica, C. juncea: witches’-broom) from China (Wang et al. 2007), and 16SrI-B (C. spectabilis, C. tetragona: ‘fasciation’ and witches’-broom) from India (Kumar et al. 2010). To our knowledge, this is the first report of the association of the phytoplasma subgroup 16SrII-D with a disease on C. pallida in India. Recent disease reports and nucleotide sequence submissions revealed that the 16SrII phytoplasmas possess a wide plant host range; particularly economically important Fabaceae pulse crops (Thorat et al. 2016). Understanding the etiology and management of little leaf disease of C. pallida is critical as this may be a potential reservoir for 16SrII phytoplasmas.

First Report of Sclerotium rolfsii Associated with Boll Rot of Cotton in India

Cotton (Gossypium hirsutum) is the most important fiber-yielding crop grown in India and worldwide. In India, during 2012-2013, 1.16 million ha were under cotton cultivation, with an average production of 480 kg/ha. As production of cotton has increased in areas, so has the occurrence of various pests and diseases. During a field survey conducted in the cotton-growing areas of Devalapura Village of the Mysore District during August and September 2013, a boll rot disease was observed with distinctive symptoms and signs of a fungal pathogen observed in 27% of fields. Incidence of the boll rot within fields ranged from 8 to 12%, while 3 to 5% stem rot was observed on boll rot-affected plants in about 15 to 18 ha of total cotton fields surveyed. White mycelial strands with spherical, dark-brown sclerotial bodies were found on the surface of infected bolls near the ground and the affected bolls were completely decayed. The disease-affected bolls were disinfested with 2% sodium hypochlorite solution for 3 min, and 5-mm samples were placed on potato dextrose agar (PDA) medium and incubated at 28 ± 2°C. The fungal colonies that grew from infected tissues were whitish with cottony aerial mycelium and abundant globoid sclerotia observed after 10 to 12 days of incubation. Sclerotial bodies were initially pale whitish, becoming dark brown over time. Based on morphological and cultural characteristics, the associated fungal pathogen was identified as Sclerotium rolfsii Sacc. The identity of the pathogen was further confirmed through PCR amplification of internally transcribed spacer (ITS) region using ITS1/ITS4 universal primer. The amplified PCR product (550 to 570 bp) was sequenced and BLASTn search comparison revealed 100% homology with Sclerotium rolfsii (Accession Nos. JF966208 and JX914480). A representative sequence of S. rolfsii was deposited in GenBank (KP412471.1 and KP412472.1). Further, pathogenicity tests were conducted on 30 healthy cotton bolls in vivo by inoculating the bolls with sclerotia obtained from 12-day-old cultures; noninoculated bolls served as controls. Characteristic boll rot symptoms were observed on 60% of total inoculated cotton bolls 7 days after inoculation and S. rolfsii was reisolated from the infected bolls and the identity of the pathogen was confirmed. S. rolfsii is a serious soil-borne fungal pathogen with a wide host range (Mullen 2001) and prevalent in tropical and subtropical regions, where high temperature and moisture are sufficient to permit growth and survival of the fungal pathogen (Punja 1985). The association of S. rolfsii with boll rot of cotton has been reported from Bangladesh (Shamsi et al. 2008). In India, major diseases of cotton include wilt (Fusarium oxysporum f. vasinfectum), anthracnose (Colletotrichum capsici), and areolate mildew (Ramularia areola) (Rangaswamy and Mahadevan 2002). S. rolfsii infect bolls on branches near ground level, causing boll decay. Numerous sclerotia produced on the affected bolls are the source of secondary inoculum and are dispersed by rain splash during the late monsoon, thus resulting in new infections on the basal stem and leaves of the cotton plant. The disease has resulted in reduced yield and market value of the crop. To the best of our knowledge, this is the first report on the occurrence of S. rolfsii causing boll rot of cotton in India.