Day :
- Workshop
Chair
Vladimir Kuzmich Chebotar
All-Russia Research Institute for Agricultural Microbiology, Russia
Co-Chair
Dr.Provorov Nikolai
Deputy Director in research of All-Russia Research Institute for Agricultural Microbiology
Session Introduction
Vladimir A Zhukov
All-Russia Research Institute for Agricultural Microbiology, Russia
Title: Molecular evolution of paralogous symbiotic receptor kinase genes in pea (Pisum sativum L)
Time : 11:20-11:50
Biography:
Vladimir A Zhukov was graduated from Saint-Petersburg State University, Faculty of Biology, Department of Genetics and Biotechnology (St. Petersburg, Russia)
and then completed his PhD at from Saint-Petersburg State University (St. Petersburg, Russia). He has worked at Aarhus University (Aarhus, Denmark) and
in CNRS (Gif-sur-Yvette, France) within the frame of joint international projects devoted to studying the genetic bases of benefi cial plant-microbe interactions.
Presently he works as the Head of the Laboratory in All-Russia Research Institute for Agricultural Microbiology (St. Petersburg, Russia). He has published more
than 20 papers in reputed Russian and international scientifi c journals.
Abstract:
In the course of interactions between legume plants and nodule bacteria, the signal molecules excreted by bacteria (Nod
factors) are specifi cally perceived by plant receptor kinases. In pea, 3 paralogous genes (Sym37, K1 and LykX) encoding
receptor kinases probably binding Nod factors are located in cluster on LG I of pea and mutations in any of these genes hamper
bacteria penetration into the plant root. In this work, the fragments of 1st exons of the genes Sym37, K1 and LykX encoding
the receptor parts of the corresponding proteins were sequenced and analyzed in 99 pea genotypes that represent virtually all
the diversity within the genus Pisum. Th e Sym37 gene sequence, according to the McDonald-Kreitman test, underwent the
pressure of positive (directional) selection. Th e K1 gene sequence possesses multiple polymorphic sites, suggesting that the
positive selection acts in favor of several allelic states of the gene. Th e sequence of the third receptor gene (LykX) possesses
the site, which seems to be critical for the functioning of the encoded protein; this site was found to be under the signifi cant
negative selection pressure. Th e assumption that these paralogous genes were diff erentiated for realization of the diverse
functions is reinforced by the results obtained. In general, the data obtained provide insights on the evolution and functioning
of symbiotic systems formed by leguminous plants.
Jacobus Johannes Marion Meyer
University of Pretoria, South Africa
Title: The role of plant growth promoting microbes in the maintenance of the mysterious fairy circles of Namibia
Time : 11:50-12:20
Biography:
Jacobus Johannes Marion Meyer is a Professor of Medicinal Plant Science at University of Pretoria, South Africa. He collaborates with several international
institutions, including the ITMO St Petersburg University.
Abstract:
The cause and maintenance of the hundreds of thousands of fairy circles in the Namibian pro-desert have not yet been
satisfactorily explained. Several diverse hypotheses have been put forward, but none have universally been accepted. We
previously found the ‘chemical footprints’ of toxic Euphorbia plants with GCMS analysis in nearly all the fairy circles of the
Garub region of Namibia. Th is implies that these toxic Euphorbia species were present, where there are fairy circles today. In
our search for the mechanism by which these toxic plants cause and maintain the fairy circles, we found that the E. damarana
extract has extremely low MIC values against plant growth promoting bacteria isolated from the ‘healthy’ soil from outside the
fairy circles. Th is indicates that the grasses will not have the benefi ts of plant growth promoting organisms when colonizing areas
where Euphorbia species have died in the harsh conditions of the Namib Desert. Th e MIC values of this extract were also found
to be very low against several human pathogenic bacteria. Pot experiments have shown that an inoculum of the rhizosphere
from grasses growing outside the fairy circles could reverse the negative eff ect of the fairy circle soil. Th is study supports the
hypothesis that the fairy circles are caused by Euphorbia spp. and maintained by the lack of plant growth promoting organisms.
Tatiana V Matveeva
St. Petersburg State University, Russia
Title: Biodiversity of naturally transgenic Linaria plants
Time : 12:20-12:50
Biography:
Tatiana V Matveeva has completed her PhD and Postdoctoral studies from the Faculty of Biology, St. Petersburg State University, Russia. She is a Doctor of
Biological Science, presently working as a Professor of the Department of Genetics and Biotechnology, Faculty of Biology at St. Petersburg State University,
Russia. She is also a Member of the Vavilov Society of Geneticists and Breeders and Member of Russian Society of Biotechnologists.
Abstract:
Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature,
insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several
species in the genera Nicotiana, Ipomea and Linaria. Th ey are called naturally transgenic plant. A sequence homologous to the
T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30
years ago and was named “cellular T-DNA” (cT-DNA). A similar cT-DNA has also been found in other species of the genus
Nicotiana. In our study T-DNA-like sequences has been detected and characterized in diff erent Linaria species, belonging to
two sections Linaria and Speciosa. In all characterized Linaria species the cT-DNA is present in two copies and organized as
a tandem imperfect direct repeat and contained the same T-DNA oncogenes and the mis gene, however there are diff erent
mutations inside the T-DNA in the investigated forms. Linaria species are medicinal transgenic plants that people used for
ages. Characterization of structure of T-DNA in close species of naturally transgenic plants is important, since they could be
good model system for study of delayed environmental risks of GMO.
Suresh Deka
Institute of Advanced Study in Science and Technology, India
Title: Application of biosurfactant for general welfare of economically important crops with special reference to management of phytopathogenic fungi
Time : 12:50-13:20
Biography:
Suresh Deka has completed his PhD in Microbial Ecology from Guwahati University in 1991 and has more than 25 years of research experience in the fi elds
of hydrocarbon degradation, bioremediation, biosurfactant and plant diseases control. He is currently working as a Professor in the Institute of Advanced Study
in Science and Technology (IASST), India. He has more than 100 publications/presentations, fi led 4 patents and produced 7 PhD. He was awarded overseas
Associateship by the Department of Biotechnology, Government of India during the year 2006-2007 and carried out Research on Biosurfactant in University of
Ulster. He has completed several research projects funded by different central governmental agencies as Principal Investigator.
Abstract:
The human population in the globe is increasing day by day. However, the land for the agriculture is decreasing with the
increasing human population. To meet the growing demands for food, agricultural production has become a matter of great
concern for all the countries. For increasing the agricultural product, agrochemicals have been used tremendously. Widespread
use of these agrochemicals cause great harm to the soil health and make the soil unproductive in the long run. To overcome the
problems, research is going on to search non-hazardous alternatives against the agrochemicals. Biosurfactant is a surface active
microbial metabolite and belongs to various classes including glycolipids, lipopeptides, fatty acids, phospholipids, neutral lipids
and lipopolysaccharides. Th e unique properties of biosurfactant have recently attracted the attention of industries to become
a possible replacement to the synthetic chemical pesticides. Biosurfactant has aspecial advantage over the chemicals being
used as they are less toxic, highly biodegradable and exhibit better environmental compatibility. Keeping this in mind, a study
was undertaken to explore the prospective application of biosurfactant isolated from native bacterial strains as an antifungal
agent against some plant pathogenic fungi. For that, a range of bacteria were isolated from hydrocarbon contaminated soil
and screened for the ability to produce biosurfactant. Th e effi cacy of the produced biosurfactants was tested in vitro as well as
in planta against some important fungal plant pathogens. Results revealed that the biosurfactant produced by the bacterium
Pseudomonas aeruginosa strain JS29 could effi ciently control the plant pathogen Colletotrichum capsici, the causal organism of
anthracnose diseases of chili and Alternaria solani, the causal organism of early blight in Tomato; P. aeruginosa strain DS9 could
control Fusarium sacchari, the causal organism of Pokkah boeng disease of sugarcane and Colletotrichum falcatum, the causal
organism of red rot of sugarcane and P. aeruginosa strain SS14 could control Fusarium oxysporum f. sp. pisi,causal organism
of Fusarium wilt of pea and Fusarium verticillioides, causal organism of stalk and ear rot of maize. Th e biosurfactants were
identifi ed as rhamnolipids. Th e rhamnolipids produced by the bacterial strains could be used to control the causal organisms
of the diseases effi ciently, which could lead to the development of an alternative, ecofriendly, cost eff ective and viable biopesticide
against these plant pathogenic fungi.
Anton Sulima
All-Russia Research Institute for Agricultural Microbiology, Russia
Title: The pea (Pisum sativum L) selectivity towards nitrogen-fi xing microsymbiont caused by receptorlike kinase gene LykX
Time : 14:00-14:30
Biography:
Anton Sulima is currently a PhD student in All-Russia Research Institute for Agricultural Microbiology, Russia. His scientifi c interests lie in the fi eld of initial stages
of legume-rhizobial symbiosis and recognition of symbiotic partners. He has been a co-author of several papers published in reputed journals and has participated
in a number of international conferences.
Abstract:
Specifi city of the symbiosis between legume plants and nodule bacteria (rhizobia) is based on ligand-receptor interactions,
during which the bacterial signal molecules (Nod-factors) are recognized by plant receptor kinases. Within the pea species,
several genotypes originating from Middle East are diff erent in their ability to perceive the Nod-factor structure, which results
in their increased selectivity for bacterial symbionts. Th is trait is controlled by plant gene Sym2 with unknown function.
We have identifi ed a new pea gene LykX which encodes a receptor-like kinase potentially capable of binding Nod-factor.
Th ere are two specifi c alleles of LykX leading to amino acid substitutions in corresponding protein which correlate with the
high selectivity in legume-rhizobial symbiosis. Th us, LykX is currently considered the most likely candidate for the Sym2.
For a further description of the role of LykX in symbiosis we performed the TILLING analysis on pea mutant collection (in
collaboration with Dr. Marion Dalmais, INRA-URGV, France). 8 mutant families with mutations presumably disrupting the
function of LykX protein (according to the in silico prediction; SIFT program) were identifi ed. Plants in each family have shown
the decreased number of nodules along with signifi cantly increased number of infection attempts. To decisively confi rm the
role of LykX, we intend to conduct the allelism test between mutant LykX alleles and wild Sym2 varieties.
Vladimir Kuzmich Chebotar
All-Russia Research Institute for Agricultural Microbiology, Russia
Title: Bacterial endophytes of grapevine (Vitis vinifera L.) as benefi cial partners for the effective plantmicrobial systems
Time : 15:00-15:30
Biography:
Vladimir Kuzmich Chebotar has completed his PhD from All-Russia Research Institute for Agricultural Microbiology. He is presently a Senior Researcher and
Head of the Lab of Microbial Technology, All-Russia Research Institute for Agricultural Microbiology. He has published more than 45 papers in reputed journals, 5
monographs and has been serving as Reviewer in a few international journals.
Abstract:
The strategy of symbiotic (cooperative) adaptations is at least as common, if not more common, in the living nature as that of
individual (autobiotic) adaptations. However, few plants have been studied in respect of their interaction with endophytic
microorganisms. Endophytic bacteria are present in all tissues and organs of grapevine but their numbers increase from shoots
to roots. B. pumilus and B. cereus have been found both in fl owers and in other parts of grapevine plants. During our study
the culturable strains of bacteria inhabiting the endosphere of grapevine cuttings of four cultivars have been isolated and
their physiological and benefi cial properties were identifi ed and characterized. Colonization process and localization sites of
introduced dsRed-labeled strain in the endosphere of grapevine have been studied using confocal scanning laser microscopy.
Th e taxonomic diversity of microorganisms isolated from the inner tissues of grapevine (four cultivars) was identifi ed based on
the analysis of the 16S rRNA gene fragments. A characteristic feature of bacterial populations of grapevine of diff erent cultivars
and diff erent geographical origin was the presence of bacteria belonging to the genus Bacillus. As a result of transformation of
several promising strains of endophytic bacteria, DsRED+phenotypes were obtained. Th eir introduction into the vegetative
parts of grapevine plants made it possible to reveal its endophytic localization of DsRED labeled pseudomonads in the vascular
tissue of the plants. Endophytic bacteria were localized in pitted vessels of grapevine shoots, single cells or in small groups.
Th e strain was mobile and could move and circulate in the vessels. So, isolated endophytic bacterial strains show biocontrol
and phytostimulating activity and may colonize internal tissues of grapevine. Th e inoculation of grapevine cuttings with these
strains before rooting may provide the plants with endophytic PGP microorganisms.
- Microbes Potential Uses|Industrial Applications of Microbes|Environmental Microbiology|Applications of Extremophiles
Chair
Choong-Min Kang
California State University, USA
Co-Chair
Eman M Khalaf
University of Guelph, Canada
Session Introduction
Choong-Min Kang
California State University, USA
Title: Reduction of feedback inhibition in homoserine kinase (ThrB) enhances the L-threonine biosynthesis
Time : 11:20-11:50
Biography:
Choong-Min Kang has completed his PhD in Microbiology from UC Davis and Postdoctoral studies from Children’s Hospital Boston, Harvard Med School. He is
currently an Associate Professor at California State University, USA. He has published more than 27 papers in reputed journals.
Abstract:
L-threonine is a limiting amino acid in livestock diets and its defi ciency results in not only malnutrition of the animals
but also releasing increased volumes of nitrogen compounds to the environment. Addition of L-threonine into livestock
feed is therefore important for healthier animals as well as a cleaner environment. Moreover, L-threonine is utilized in both
pharmaceutical and cosmetic industries. Currently, L-threonine is produced by E. coli, which makes the purifi cation of
L-threonine diffi cult because it produces endotoxins. Th us, we seek to over-produce L-threonine by using Corynebacterium
glutamicum, a GRAS (generally regarded as safe) microorganism. C. glutamicum produces L-threonine from aspartate
through an enzymatic pathway involving aspartate kinase (LysC), aspartate semialdehyde dehydrogenase (Asd), homoserine
dehydrogenase (Hom), homoserine kinase (Th rB) and threonine synthase (Th rC). Among these, LysC, Hom and Th rB are
feedback inhibited by the end-product, L-threonine. Releasing the feedback inhibition in LysC and Hom through mutating their
allosteric site has successfully increased L-threonine biosynthesis. However, it has been unsuccessful to remove the feedback
inhibition in Th rB because L-threonine inhibits the enzyme by competing with L-homoserine (substrate) for the same active
site. To genetically separate the catalytic activity and the feedback inhibition in Th rB of C. glutamicum, we mutated a residue
at the gate of the active site into various amino acids such as hydrophobic residues (Leu and Val) and hydrophilic residue (Ser
and Gly). Enzymatic kinetics with the wild-type and mutant forms of Th rB showed that one specifi c mutation increased Ki
for L-Th reonine about 5-fold while it increased Km for homoserine only 2-fold. More importantly, when we introduced this
mutation into C. glutamicum and E. coli, it increased approximately 20% of L-threonine production. Our approach can be
applied to other metabolic enzymes that are similarly regulated by competitive feedback inhibition.
Rajesh Sani
South Dakota School of Mines and Technology, USA
Title: Extremophiles in biofuel synthesis from solid wastes
Time : 11:50-12:20
Biography:
Rajesh Sani is an Associate Professor in the Department of Chemical and Biological Engineering at South Dakota School of Mines and Technology, USA. His
research includes extremophilic bioprocessing of lignocellulose-based renewable for biofuels and bioproducts and bioprospecting of extremophilic microorganisms
for developing more effi cient and cost-effective biofuel (bioenergy) production technologies. Over the past 10 years, he has been the PI or Co-PI on over $10.5
million in funded research. He has one patent, fi ve invention disclosures and published 50 peer-reviewed articles in high impact factor journals and has contributed
in several book chapters. In addition, he has been a proposal Reviewer and Panelist for the Federal Agencies and also serves the Industrial Microbiology profession
as “Biocatalysis Program Committee Member” of the Society for Industrial Microbiology and Biotechnology (SIMB), Technical Session Chair at the Annual American
Institute of Chemical Engineers (AIChE) and SIMB and is also an Associate Editor.
Abstract:
More than 34% of a typical solid waste are food, wood and yard waste whereas paper and paperboard contribute to another
25% to the ever increasing issue of waste management and its disposal. To date physiochemical pretreatment of solid
wastes has been shown a necessary step for previously listed consolidated biological processes which increases the overall cost
of the process e.g., plant biomass is inexpensive ($2-4/GJ at a cost of $39-60/dry ton biomass) but its pretreatment cost ($15-25/
GJ) dramatically reduce the overall cost-effi ciency of the process. An alternative to the bioprocess involving pretreatment is the
development of an effi cient and cost-eff ective single step process for untreated solid waste management using extremophiles.
For example, thermophiles and their enzymes can play important roles in many kinds of bioprocessing including in conversion
of non-food biomass into biofuels. Th e Homestake gold mine (8000 ft . deep, Lead, SD) off ers a unique opportunity for direct
exploration of the deep biosphere environment. Using soil/biofi lm samples of deep biosphere of the Homestake Gold Mine,
compost facility (Rapid City, SD) and Hot Springs State Park (Th ermopolis WY), we have isolated several thermophilic
cellulose and xylan-degrading and fermenting pure cultures belonging to the genera Brevibacillus, Paenibacillus, Clostridium,
Bacillus and Geobacillus. Unique characteristics of lignocellulose-deconstructing enzymes produced by mine and compostthermophiles
include optimum temperatures of >70ºC, pH ranges from 4-8 and high thermostability (e.g., at 60ºC, 50%
of cellulases and xylanases activities were retained in 35 and 23 days of incubation, respectively). Our thermophiles also
produced biohydrogen or bioethanol in a single step bioprocessing of various inexpensive regional untreated biomass (e.g.,
prairie cord grass and corn stover). Th is talk will describe the limitations in exiting solid waste conversion technologies and
possible ways to overcome those limitations using thermophiles and their enzymes. Th e infl uence of high temperatures on
various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis
and fermentation, simultaneous saccharifi cation and fermentation and extremophilic consolidated bioprocess will discussed.
Integrated decentralized thermophilic biofuel production employing cellulolytic and fermentative thermophiles in a single
step consolidated process will also be discussed.
Eman M Khalaf
University of Guelph, Canada
Title: Taxonomic diversity of cucurbit seed associated endophytes
Time : 12:20-12:50
Biography:
Eman M Khalaf has completed her BSc and PhD from Pharmacy School, Mansoura University, Egypt. She is presently working as a Lecturer of Microbiology,
Pharmacy School, Damanhour University, Egypt. She has spent two years as a Postdoctoral fellow in Raizada Lab (Benefi cial Microbes Lab), University of Guelph,
Canada
Abstract:
Endophytes are potentially benefi cial microbes that live inside plants and can be utilized as biofertilizers and biocontrol
agents by promoting plant growth and/or suppressing phytopathogens. Few endophytes have previously been isolated
from the cucurbit plant family which includes economically important crops including watermelon, cucumber, cantaloupe,
pumpkin and squash. In the present study, 169 bacterial endophytes and 10 fungal endophytes were isolated from seeds of
diff erent varieties of diverse cucurbits; the most comprehensive characterization of endophytes from this plant family. 16S
rDNA and internal transcribed spacers (ITS4 and ITS1F) fi ngerprinting were used to taxonomically classify the isolated
bacteria and fungi, respectively. Phylogenetic analysis of bacterial isolates showed Bacilli was the most abundant class including
six diff erent bacterial families. Th e γ-proteobacteria and Actinobacteria were also represented in four diff erent families. Bacillus
was the core microbiota that was conserved across tested cucurbits genera. Strains of the genus Paenibacillus were exclusively
isolated from Cucumis sativus L, Cucumis melo L and Cucurbita pepo L. var pepo L. Th ree diff erent genera of lactic acid bacteria
(LAB) were recovered from C. sativus L, C. melo L., Citrullus lanatus var. lanatus and Cucurbita pepo L var. turbinata. Our
results suggest co-evolution of seed microbial consortia with their host plants. We now propose to test these novel endophytic
microbial strains for their ability to suppress important fungal and bacterial pathogens of the cucurbit family.