Bacterial community analysis on Sclerotium-suppressive soil.

Difficulties in controlling the soil-borne plant pathogenic fungus Sclerotium rolfsii favoured the analysis of its suppressive soil for better understanding. In the present study, culture-independent molecular technique was used to analyse the bacterial communities of suppressive soil and conducive soil. Hence, metagenomic DNAs from both kinds of soils were directly extracted and their sequence polymorphism was analysed by targeting hypervariable domains, V4 + V5, of the 16S rRNA gene. The results of 16S rRNA gene-driven bacterial community diversity analysis along with soil physicochemical and biological properties clearly discriminated S. rolfsii suppressive soil from conducive soil. The dominant phylogenetic group of suppressive soil is Actinobacteria followed by Proteobacteria. The other groups include Acidobacteria, Firmicutes and Cyanobacteria. In contrast, conducive soil had very few Actinobacterial sequences and was dominated by Gamma- and Betaproteobacteria. Based on the relative proportion of different bacterial communities, their diversity and species richness were observed more in suppressive soil than in conducive soil. The present study identifies the dominant bacterial community which shares S. rolfsii suppressiveness.

Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms.

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector-leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890 bp. From this amplified product, specifically a target of 69 bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9 × 105 g of tissue) followed by roots (7.4 × 105 g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

Effect of culture media and environmental factors on mycelial growth and pycnidial production of Lasiodiplodia theobromae in physic nut (Jatropha curcas).

Physic nut (Jatropha curcas) is an important commercial bio-diesel plant species and is being advocated for development of waste and dry land. The collar and root rot caused by Lasiodiplodia theobromae is an important soil borne disease which causes considerable yield loss in this crop. In this study, the effects of culture media, temperature, photoperiod, carbon and nitrogen sources and pH on mycelial growth and pycnidial production were evaluated. Among the growth media tested, potato dextrose agar supported the highest growth followed by potato sucrose agar and corn meal agar. Among several carbon sources tested, carboxy methyl cellulose and sucrose were found superior for growth and pycnidial production. The nitrogen sources viz., ammonium oxalate and ammonium dihydrogen phosphate were recorded maximum mycelial growth and pycnidial production. The fungus grows at pH 5.0-9.0 and optimum growth was observed at pH 7.0.

Host-Specific Toxin Production by Rhizoctonia solani, the Rice Sheath Blight Pathogen.

ABSTRACT Rhizoctonia solani, the rice sheath blight pathogen, produces a toxin that reproduces all symptoms of the disease. The toxin has been partially purified and it was found to be a carbohydrate containing glucose, mannose, N-acetylgalactosamine, and N-acetylglucosamine. The toxin was also detected in infected leaves. Highly virulent isolates produced more toxin than less virulent isolates. Several R. solani isolates from rice and one each from cotton and tomato produced a similar toxin. All rice cultivars tested were susceptible to the pathogen and sensitive to the toxin. Host specificity of the toxin has been demonstrated using hosts and nonhosts of the pathogen.

Antifungal activity of chitinases produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane.

Chitinase production and growth of certain fluorescent pseudomonads isolated from sugarcane rhizosphere on different subtrates were studied. When chitin was substituted for glycerol in King's B medium, 3 of the 4 strains showed enhanced bacterial multiplication. Bacterial cells grown on chitin-containing medium showed enhanced antifungal activity against Colletotrichum falcatum Went causing red rot disease in sugarcane. Chitinase production was significantly higher when chitin was amended to King's B medium. Higher chitinase production was also recorded when fluorescent pseudomonad strains were grown in the medium containing crab-shell chitin. Cell-free bacterial culture filtrate from chitin-containing medium significantly inhibited mycelial growth of the pathogen. These cell-free conditioned media contained 3 to 7 polypeptides. Western blot analysis revealed five isoforms of chitinase with molecular masses of 47, 36, 32, 20 and 18.5 kDa. A possible role of chitinases in red rot disease management is discussed.

Two pathogenesis-related peroxidases in greengram (Vigna radiata (L.) wilczek) leaves and cultured cells induced by Macrophomina phaseolina (Tassi) Goid. and its elicitor.

An elicitor has been isolated from Macrophomina phaseolina, the root rot and leaf blight pathogen of greengram. Suspension-cultured cells of greengram were established which responded to the fungal elicitor. When greengram leaves were inoculated with M. phaseolina two new peroxidases appeared. Similarly, two new peroxidases could be detected in suspension-cultured greengram cells when treated with the fungal elicitor. These peroxidases were purified by column chromatography and their molecular masses were 27 and 38 kDa. The new peroxidases detected in both leaves and cultured cells appear to be similar with the same molecular weights.

Immunology of the pathogen virulence and phytotoxin production in relation to disease severity: a case study in sheath blight of rice.

Polyclonal antibodies against purified Rhizoctonia solani toxin obtained from infected rice sheath tissues (sheath blight toxin, SBT) and culture filtrates (culture filtrate toxin, CFT) were developed in rabbit and chicken. The IgG was isolated from serum and egg yolk of rabbit and chicken, respectively, and their specificity was investigated by indirect ELISA. Antibodies developed against CFT and SBT in rabbits exhibited relatively higher titer values when compared to chicken antibodies. Positive correlation was observed between the degree of sheath blighting and the levels of antigens induced by each isolate during sheath blight symptom development as detected by rabbit SBT antibody and the isolate RS7 was identified as most virulent. Optimization of incubation period for maximum toxin production in liquid medium and rice sheaths indicated that the production of CFT and SBT is maximum after 15 d and 6 d of pathogen inoculation. Studies of the possible translocation of RS-toxin in rice plants upon inoculation with R. solani showed downward translocation as detected by rabbit/chicken SBT antibodies. Since plant inoculation required a higher concentration of inoculum and maintenance of plants, serological assay by ELISA is more sensitive than whole-plant assays in detecting RS-toxin, with the advantage that ELISA also allows rapid determination of RS-toxin production.

Induction of phenylpropanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato.

Pseudomonas fluorescens (two native strains, one collection strain and their strain mixtures in all possible combinations) when applied through seed, seedling dip, soil and on leaf significantly reduced the tomato spotted wilt virus (TSWV) disease. In P. fluorescens-treated plants, the peroxidase and phenylalanine ammonia-lyase activity increased. Accumulation of phenolic compounds and lignin were shown to be increased in the P. fluorescens-treated plants. Isoperoxidase native PAGE indicated that the peroxidase isoforms in tomato plants induced by fluorescent pseudomonads were different from the control plants; this suggests that the general phenylpropanoid pathway is probably stimulated in tomato plants treated which in turn led to significant reduction in TSWV.

Risk assessment of insecticides used in rice on miridbug, Cyrtorhinus lividipennis Reuter, the important predator of brown planthopper, Nilaparvata lugens (Stal.).

The green miridbug, Cyrtorhinus lividipennis, an important natural enemy of the rice brown planthopper (BPH), Nilaparvata lugens plays a major role as a predator in suppressing the pest population. The study assessed the impact of certain potential insecticides used in the rice ecosystem on the miridbug predator and brown planthopper through contact toxicity. Eleven insecticides, including neonicotinoids, diamides, azomethine pyridines, carbamates, pyrethroids, organophosphates and cyclodienes were selected to test their toxicities against the nymphs of C. lividipennis and N. lugens. Median lethal concentration (LC(50)) was determined for each insecticide using an insecticide-coated vial (scintillation) residue bioassay, which revealed BPMC as the highly toxic chemical with an LC(50) of 0.003mga.iL(-1) followed by ethofenprox and clothianidin with LC(50) of 0.006mga.iL(-1) at 48 HAT against C. lividipennis and ethofenprox as the highly toxic chemical with an LC(50) of 0.009mga.iL(-1) followed by clothianidin with an LC(50) of 0.211mga.iL(-1) at 48h after treatment (HAT) against N. lugens. Among the insecticides tested, the cyclodiene compound, endosulfan had the lowest acute contact toxicity (LC(50)=66.65mga.iL(-1) at 48 HAT) to C. lividipennis. Among the insecticides tested, endosulfan, chlorpyriphos, acephate and methyl parathion are regarded as safer to C. lividipennis based on selectivity ratio, hazard quotient and probit substitution method of risk assessments.

Molecular biology of Ganoderma pathogenicity and diagnosis in coconut seedlings.

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10-13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.

ACC deaminase from Pseudomonas fluorescens mediated saline resistance in groundnut (Arachis hypogea) plants.

AIM: To study the effect of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase from Pseudomonas fluorescens against saline stress under in vitro and field conditions in groundnut (Arachis hypogea) plants. METHODS AND RESULTS: Four plant growth-promoting rhizobacteria (PGPR) strains were used in this study to evaluate their efficacy in groundnut plants against saline stress under in vitro. Among the four PGPR strains used, Ps. fluorescens strain TDK1 showed greater performance in improving the plant growth parameters of groundnut seedlings in vitro. PCR amplification using Pseudomonas-specific 16S-23S rRNA internal transcribed spacers (ITS) primers revealed that all the four strains belonged to the group of fluorescent pseudomonads. ITS region of Ps. fluorescens strain TDK1 was cloned and sequenced. ACC deaminase activity using biochemical and molecular (PCR) analysis revealed that among all the four strains, Ps. fluorescens strain TDK1 showed greater amount of ACC deaminase activity and positive reaction to PCR amplification. ACC deaminase gene from Ps. fluorescens strain TDK1 was isolated, cloned and sequenced. Pseudomonas bioformulations were developed and they were tested in groundnut plants under saline-affected soils. The results indicated the superior performance by Ps. fluorescens strain TDK1 possessing ACC deaminase activity in improving yield parameters in groundnut plants despite salinity. CONCLUSIONS: Pseudomonas fluorescens strain TDK1 possessing ACC deaminase activity enhanced the saline resistance in groundnut plants, which in turn resulted in increased yield when compared with the groundnuts treated with Pseudomonas strains not having ACC deaminase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The promising role of ACC deaminase from Ps. fluorescens strain TDK1 in alleviating saline stress has been concluded in groundnut plants. This study will be useful for exploiting the activity of ACC deaminase from microbial strains against various biotic and abiotic stresses wherever ACC accumulated as precursor for ethylene biosynthesis.

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight.

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.

Purification and characterization of an extracellular alpha-glucosidase protein from Trichoderma viride which degrades a phytotoxin associated with sheath blight disease in rice.

AIMS: To purify and characterize an extracellular alpha-glucosidase from Trichoderma viride capable of inactivating a host-specific phytotoxin, designated RS toxin, produced by the rice sheath blight pathogen, Rhizoctonia solani Kühn. METHODS AND RESULTS: The host-specific RS toxin was purified from both culture filtrates (culture filtrate toxin, CFTox) and R. solani-inoculated rice sheaths (sheath blight toxin, SBTox). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses of extracellular proteins, purified from a biocontrol fungus T. viride (TvMNT7) grown on SBTox and CFTox separately, were carried out. The antifungal activity of the purified high molecular weight protein (110 kDa) was studied against RS toxin as well as on the sclerotial germination and mycelial growth of R. solani. Enzyme assay and Western blot analysis with the antirabbit TvMNT7 110-kDa protein indicated that the protein was an alpha-glucosidase. The 110-kDa protein was highly specific to RS toxin and its Michaelis-Menten constant value was 0.40 mmol l-1 when p-nitrophenyl alpha-D-glucopyranoside was used as the substrate. The isoelectric point of the protein was 5.2. N-terminal sequencing of the alpha-glucosidase protein showed that its amino acid sequence showed no homology with other known alpha-glucosidases. CONCLUSION: This appears to be the first report of the purification and characterization of an alpha-glucosidase capable of inactivating a host-specific toxin of fungal origin. The alpha-glucosidase is specific to RS toxin and is different from the known alpha-glucosidases. SIGNIFICANCE AND IMPACT OF THE STUDY: As RS toxin could be inactivated by the microbial alpha-glucosidase enzyme, isolation of the gene that codes for the enzyme from T. viride and transfer of the gene to rice plants would lead to enhanced resistance against sheath blight pathogen by inactivation of RS toxin.

Inactivation of phytotoxin produced by the rice sheath blight pathogen Rhizoctonia solani.

The rice sheath blight pathogen, Rhizoctonia solani, produces a toxin designated as RS-toxin, a carbohydrate compound containing mainly alpha-glucose and mannose. Different microflora were tested for RS-toxin inactivation. Isolates of Trichoderma viride inactivated this toxin when it was provided as the sole food source, and these isolates reduced the severity of toxin-induced symptoms and electrolyte leakage from rice cells. The best-performing isolate, TvMNT7, produced two extracellular proteins of 110 and 17 kDa. The high molecular mass protein was shown to have alpha-glucosidase activity. The purified 110 kDa protein was able to reduce RS-toxin activity.