Intra-regional diversity of rice bacterial blight pathogen, Xanthomonas oryzae pv. oryzae, in the Andaman Islands, India: revelation by pathotyping and multilocus sequence typing.

AIM: To investigate the genetic and pathogenic variability of Xanthomonas oryzae pv. oryzae causing bacterial blight in rice on the remote Andaman Islands, India. METHODS AND RESULTS: A total of 27 yellow-pigmented bacterial isolates representing rice fields of Andaman Islands incited blight on the susceptible-rice cultivar, C14-8. Phenotypic, pathogenic traits and 16S rRNA gene sequences revealed their identity as X. oryzae pv. oryzae. Virulence profiling indicated the prevalence of seven pathotypes of X. oryzae pv. oryzae on the Islands. Pathotypes-VI and -VII were highly virulent, whereas the pathotype-I was less virulent. Multilocus sequence typing based on nucleotide sequence polymorphism in nine housekeeping genes dnaK; fyuA; gyrB (two loci): rpoD; fusA; gapA; gltA and lepA clustered 27 isolates into 17 sequence types (STs) segregated into two clonal-complexes (CC). While CC-I comprised of isolates from Andaman Island, the CC-II is a mixture of isolates representing mainland India and Andaman Island. The data revealed trans-boundary pathogen introduction and a consequent intra-regional diversification on these islands due to the deployment of different rice cultivars in different regions. CONCLUSIONS: Genotyping and pathotyping of sland isolates revealed seven pathotypes distributed in two clonal complexes with strong indications for trans-boundary movement and consequent diversification of the bacterial pathogen. Highly virulent pathotypes of X. oryzae pv. oryzae that could overcome combinations of R-genes, xa13+Xa21 as well as xa5+xa13 were found prevalent in the Andaman Islands SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic and virulence analysis of X. oryzae pv. oryzae in the Andaman Islands revealed introduction and host-mediated regional diversification and local adaptation of X oryzae pv. oryzae. The study calls for the need of multi-gene pyramiding for durable disease resistance and establishing stringent quarantine measures for safeguarding island agricultural practices in the future.

Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees.

The effectiveness of aqueous extracts of various medicinal plants in detoxification of aflatoxin B1 (AFB1) was tested in vitro by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Among the different plant extracts, the leaf extract of Vasaka (Adhatoda vasica Nees) showed the maximum degradation of AFB1 (≥ 98%) after incubation for 24h at 37 °C. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly reduced by heating to 100 °C for 10 min or autoclaving at 121 °C for 20 min. Dialysis had no effect on aflatoxin detoxifying ability of A. vasica extract and the dialyzed extract showed similar level of detoxification of AFB1 as that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% of the toxin was degraded within 6h and ≥ 95% degradation was observed after 24h of incubation. Detoxification of AFB1 by A. vasica extract was further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis. Phytochemical analysis revealed the presence of alkaloids in methanolic extract of A. vasica leaves. A partially purified alkaloid from A. vasica leaves by preparative TLC exhibited strong AFB1 detoxification activity.

Characterization of betasatellite associated with the yellow mosaic disease of grain legumes in Southern India.

Yellow mosaic disease caused by mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus (the family Geminiviridae) is a major constraint in cultivation of grain legumes in India. The urdbean (Vigna mungo (L.) Hepper) and mungbean (Vigna radiata (L.) R. Wilczek) samples affected with yellow mosaic disease exhibits yellow mosaic symptoms along with leaf puckering and leaf distortion in Tamil Nadu. Hence the study was performed to find out if there was any association and influence of betasatellite DNA on the symptom expression of MYMV. Full length viral clones of DNA A and DNA B were obtained through rolling circle amplification from YMD infected samples and identified as mungbean yellow mosaic virus. Interestingly, betasatellite was found to associate with MYMV, and its nucleotide sequence analysis showed its 95% identity with papaya leaf curl betasatellite (DQ118862) from cowpea. The present study represents the first report about the association of papaya leaf curl betasatellite with MYMV and represents a new member of the emerging group of bipartite begomovirus associated with betasatellite DNA.

Detoxification of oxalic acid by pseudomonas fluorescens strain pfMDU2: implications for the biological control of rice sheath blight caused by Rhizoctonia solani.

Rhizoctonia solani isolates varying in their virulence were tested for their ability to produce oxalic acid (OA) in vitro. The results indicated that the virulent isolates produced more OA than the less virulent isolates. In order to isolate OA-detoxifying strains of Pseudomonas fluorescens, rhizosphere soil of rice was drenched with 100 mM OA and fluorescent pseudomonads were isolated from the OA-amended soil by using King's medium B. These isolates were tested for their antagonistic effect towards growth of R. solani in vitro. Among them P. fluorescens PfMDU2 was the most effective in inhibiting the mycelial growth of R. solani. P. fluorescens PfMDU2 was capable of detoxifying OA and several proteins were detected in the culture filtrate of PfMDU2 when it was grown in medium containing OA. To investigate whether the gene(s) involved in OA-detoxification resides on the plasmids in P. fluorescens PfMDU2, a plasmid-deficient strain of P. fluorescens was generated by plasmid curing. The plasmid-deficient strain (PfMDU2P-) failed to grow in medium containing OA and did not inhibit the growth of R. solani. Both PfMDU2 and PfMDU2P- were tested for their efficacy in controlling sheath blight of rice under greenhouse conditions. Seed treatment followed by soil application of rice with P. fluorescens strain, PfMDU2, reduced the severity of sheath blight by 75% compared with the control, whereas PfMDU2P- failed to control sheath blight disease.

Induction of resistance in host against the infection of leaf blight pathogen (Alternaria palandui) in onion (Allium cepa var aggregatum).

The Pseudomonas fluorescens isolate Pfl was found to inhibit the growth of pathogen Alternaria palandui, in vitro. In the present study, foliar application of a talc-based formulation of Pfl significantly reduced the incidence of leaf blight of onion, caused by A. palandui. Induction of defense-related proteins viz., chitinase, beta-1,3 glucanase, peroxidase (PO) and polyphenol oxidase (PPO) by application of Pfl, was studied against A. palandui infection in resistant (IHR 56) and susceptible (MDUI) onion cultivars. Chitinase in both cultivars, with or without challenge-inoculation of A. palandui revealed changes in the isoform pattern. The Native-PAGE of PO showed induction of PO2 isoform in both the cultivars, in response to inoculation of pathogen. Isoform analysis of PPO also exhibited induction in the Pfl-treated plants challenged with pathogen. Similarly, the activity of beta-1,3-glucanase was greatly induced in Pfl-treated plants, challenged with pathogen as compared to controls. Thus, the P. fluorescens-treated plants showed significant increase in the levels of the defense enzymes, in comparison to the plants challenged with the pathogen.

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen.

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.

Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars.

Genetic transformation has been attempted for management of rice sheath blight disease, caused by Rhizoctonia solani. We introduced a PR-3 rice chitinase gene (RC7), isolated from R. solani-infected rice plants, into indica rice cultivars IR72, IR64, IR68899B, MH63, and Chinsurah Boro II by the biolistic and PEG-mediated transformation system. Inheritance was studied up to the T(2) generation by Southern blot analysis. Western blot analysis of transgenic plants with polyclonal antibody revealed the presence of chitinase protein with a molecular weight of 35 kDa that reacts with chitinase antibody. The transformants synthesized different levels of chitinase proteins constitutively and progeny from the plants containing the chitinase gene showed different levels of enhanced resistance when challenged with the sheath blight pathogen R. solani.

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.