M. Y. Sreenivasa

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.

Antifungal activity of Azotobacter nigricans against trichothecene-producing Fusarium species associated with cereals

Antifungal efficacy of Azotobacter nigricans on Fusarium infection, total seedlings mass, root and shoot length, and seed germination in maize, sorghum, and wheat were investigated. Antifungal efficacy of A. nigricans against Fusarium sporotrichioides, Fusarium graminearum, Fusarium poae and Fusarium equiseti showed a significant reduction in growth and Fusarium infection incidence (up to 50%) in all the three treated cereals. However, challenge inoculation of Fusarium spp. to the three cereals showed 100% infection incidence. Total mass of the maize seedlings increased two fold by A. nigricans treatment; however, only a slight increase was observed in sorghum and wheat seedlings. The highest vigour index recorded in maize was 1321 against Fusarium crookwellense, 1616.71 against Fusarium sporotrichioides in sorghum, and 1584.8 against Fusarium acuminatum in wheat treated with A. nigricans. Highest germination incidence of 64% was in maize, 67% in sorghum, and 56% in wheat treated with A. nigricans.

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.

Molecular Identification of Chrysanthemum chlorotic mottle viroid Infecting Chrysanthemum in Karnataka, India

Chrysanthemum chlorotic mottle viroid (CChMVd, genus Pelamoviroid, family Avsunviroidae) has been reported to infect chrysanthemum (Dendranthema x grandiflorum). CChMVd was first reported in 1967 in the cultivar Yellow Delaware in New York State, USA (Dimock et al. 1971). Presence of CChMVd has previously been recorded, based on the symptoms and bioassay, in India (Singh et al. 1978). Karnataka State of India is the prominent chrysanthemum-growing region, with 19,161 ha cultivated under this crop and accounting for more than 40% of country’s flower production. During late summer 2010 (May and June), chrysanthemum leaves with yellow-green mottling and chlorotic spots were noted, indicating the possible association with CChMVd in Karnataka. Total RNA was extracted using a 2% CTAB buffer from leaf samples of symptomatic plants (Adkar-Purushothama et al. 2013). For the detection of CChMVd, reverse transcription PCR (RT-PCR) was performed using PIII/PIV as described previously (De la Pena et al. 1999). PCR products of the expected sizes (∼398 to 401 bp) were cloned and sequenced. BLAST analysis of the obtained sequences confirmed the presence of CChMVd in Karnataka. In order to estimate the incidence of CChMVd, the same RT-PCR assay was performed on 80 randomly selected chrysanthemum leaf samples of four different cultivars (Redgold, Bangalore, Sarval, and CO-1) collected from four chrysanthemum-growing districts of Karnataka. A PCR product of the size expected for CChMVd was detected in 8 of 80 samples analyzed, accounting for the infection incidence of 10%. To identify the prominent CChMVd variants, 10 clones obtained from the four representative samples were sequenced. To confirm the sequence of the primer regions, an additional set of primers CCh1/CCh2 were used for RT-PCR (Hosokawa et al. 2005) with high-fidelity LA-Taq polymerase (Takara-Bio, Japan). Alignment of all 40 sequences revealed the presence of at least two major sequence variants of CChMVd: CChMVd-Ind-1 (GenBank Accession No. KP262531) and CChMVd-Ind-3 (KP262533). Both the sequence variants had UUUC sequences (nucleotide position 82 to 85) in the tetraloop of the CChMVd, which is often associated with induction of symptoms in susceptible chrysanthemum cultivars (De la Pena et al. 1999). CChMVd-Ind-1was found to differ from CChMVd-Ind-3 by 17 nucleotide substitutions. BLAST analysis of CChMVd-Ind-1 showed 98% sequence identity with the CChMVd strain reported from Spain (GenBank Accession No. FJ647515) and from Himachal Pradesh state of India (FN646404), whereas CChMVd-Ind-3 showed 96% sequence identity with CChMVd strain W2-4, isolated from China (HQ891014). To our knowledge, this is the first molecular evidence for the presence of CChMVd infecting chrysanthemum in Karnataka State, India. The high degree of sequence diversity reported in the Karnataka isolates of CChMVd will help to clarify the evolution of this viroid and its possible threat to important crops.

One pot synthesis of thiazolo[2,3-b]dihydropyrimidinone possessing pyrazole moiety and evaluation of their anti-inflammatory and antimicrobial activities

A series of pyrazole integrated thiazolo[2,3-b]dihydropyrimidinone derivatives were synthesized as dual anti-inflammatory and antimicrobial agents. Among the compounds studied, 3-fluoro-4-methylphenyl analogues (3a, 3e, and 3i) are considered to be promising leads for novel anti-inflammatory agents compared with the standard drug. The superior antimicrobial property of the compounds 3a, 3b, and 3d indicates that 3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazole substitution is a favourable site for high activity. Molecular docking studies were carried out in order to predict the hypothetical binding mode of these compounds to the COX-2 isoenzyme. The results of the present study suggest that 1,3-diaryl pyrazole substitution on thiazolo[2,3-b]dihydropyrimidinone derivatives might potentially constitute a novel class of anti-inflammatory agents with antimicrobial property and could be an interesting approach for the design of new selective COX-2 inhibitory agents.

Nested PCR Method for Early Detection of Fumonisin Producing Fusarium verticillioides in Pure Cultures, Cereal Samples and Plant Parts

ABSTRACT Fumonisin-producing Fusarium verticillioides was detected in cereal samples by semi-nested PCR using one forward, VERTF-1, and two reverse primers, VERTR and VERTF-2. A total of 326 Fusarium species isolated from maize, paddy, sorghum, wheat and pearl-millet samples were subjected to a first round of n-PCR with the species specific VERTF-1 and VERTR set of primers which recorded 59.50% of F. verticillioides. Further, second round of n-PCR scored 53.98% of fumonisin-producing F. verticillioides with fumonisin specific VERTF-1 and VERTF-2 set of primers. Maize samples recorded the highest frequency of fumonisin-producing F. verticillioides 40.98%, followed by paddy 33.33%, and sorghum 12.50%. Sensitivity of nested PCR was conducted by whole grain experiment of the cereals, roots and leaves of the cereals by diluting the DNA 10-100 times, in which 1:50, 1:75, and 1:100 diluted samples recorded positive. This method can be used for the early detection of fumonisin producing F. verticillioides occurring on cereals.

Affordable and reliable plant sap-mediated template preparation for the detection of various phytopathogens by PCR assay

Plant pathogens like ‘Ca. Liberibacter’, phytoplasma, viruses and viroids cause diseases to almost all economically important plants. A simple and fast sap-mediated polymerase chain reaction (PCR) method for the detection of these pathogens infecting various plant hosts is described in the present study. Sap from selected plants was drawn aseptically on parafilm, from the mid-rib of young leaves. Depending on the type of host plant, sap was diluted to optimal concentration before PCR analysis. ‘Ca. Liberibacter’, Citrus tristeza virus (CTV) and Citrus exocortis viroid (CEVd) infecting citrus, and ‘Ca. Phytoplasma’ infecting pepper and sandal trees were tested by sap-mediated PCR. The reliability of this procedure was evaluated by comparing the findings with previously described protocols. The sap-mediated nucleic acid template preparation for PCR assay is devoid of laborious nucleic acid extraction and expensive chemicals. Hence the present method is rapid, economical and so can be employed for diagnosis of large number of plant samples.

Evaluation of efficiency of hemi-nested PCR assay for the detection of ‘Candidatus Liberibacter’ infecting citrus

The present study reports the development and evaluation of a hemi-nested polymerase chain reaction (hnPCR) assay for the efficient detection of ‘Candidatus Liberibacter’ infecting citrus plants. A set of new primers was designed by alignment of nucleotide sequences of the β-operon (rplKAJL-rpoBC) ribosomal protein genes from all the known ‘Candi-datus Liberibacter asiaticus’ isolates reported in Genbank. Hemi-nested PCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from hemi nested-PCR reactions confirmed the specificity of new primer pairs for ‘Candidatus Liberibacter asiaticus’. The reliability and sensitivity of hnPCR was evaluated by comparing it to real-time PCR.

Comparative studies on the changes of total soluble proteins and protease activity in Georgian commercial peanuts contaminated by Aspergillus flavus

The presence of Aspergillus species is an indicator of storage conditions, which also suggests the possibility of several biochemical changes in grains. A comparative change in total soluble proteins and protease activity was determined in commercial peanut seeds collected from Georgia State. Protein contents of healthy peanuts, naturally contaminated peanuts and then artificially inoculated peanut seeds with A. flavus were estimated by Bradford method, and protease activity was also determined by using the Protease Detection Kits. Protein contents and the protease activity of the peanuts varied from sample to sample. The soluble protein content of seeds was significantly higher in healthy peanuts than in artificially inoculated or naturally infected peanuts with A. flavus. Protease activity was found to be higher in artificially inoculated seeds than in either naturally infected or healthy peanuts. Level of soluble proteins in buffer extracts of contaminated seeds decreased with incubation time, and protease activity increased with incubation time. These changes may be attributed to host response due to infection, contribution by A. flavus or due to biochemical alterations that occur naturally during the transition from endosperm to seedling during incubation period.