 |
|
|
|
California State University Northridge Biology 470 - Biotechnology |
||
|
|
|
|
California State University Northridge Biology 470 - Biotechnology |
||
Lecture 9
Microbial-Plant Relationships
What do you need for a good crop?
Genetic make-up of the plant
Hold that thought - we'll be talking about this later
Correct balance of minerals in soil
Resource reading on Nutrient Stress: http://faculty.washington.edu/lindacs/EHUF%20478/Nutrient%20stress.html
One "low-tech" way that this has been controlled is by crop rotation. Rotation of soybeans and red clover (also known as "green manure"), for example, can restore nitrogen to the soil while keeping application costs down, prevent propagation of diseases from year to year, and prevent erosion. This is a complicated matter that also depends on economic interests. See for example, these data from Tony Vyn, Crop Science Department, University of Guelph:
|
Source: http://www.gocorn.net/rotation2.html A GOOD CROP ROTATION - YOU CAN'T
FARM WITHOUT IT Ontario soybean acreage exceeded corn acreage by
approximately 40% in 1997. The doubling of soybean acreage in the last 10 years has
resulted in dramatic shifts in crop rotation practices on Ontario farms. Many farms
which 20 years ago had rotations involving 2 or more consecutive years of corn, now
have rotations which contain 2 or more consecutive years of soybeans. Many cash-crop
farmers have also dropped winter wheat from the 3-year rotation system of corn-soybean-wheat
which was popular during the 1980's. Aside from soybean disease concerns, the two
biggest risks with increasing the frequency of soybeans in crop rotations are (a)
loss of soil structure and (b) limiting potential corn and soybean yields. Ontario's rotation research has clearly indicated increased corn yields when corn is rotated with other crops. Highest yields occurred when forage legumes (i.e. alfalfa or red clover) precede corn in rotation. Research conducted by Doug Young of Ridgetown College observed corn yield responses to rotation were up to 30% higher than continuous corn on medium and fine-textured soils in Southwestern Ontario (Table 1).
was not plowed under but harvested for seed the following year and then fall plowed. * All corn treatments were fertilized with 160 lbs/ac of N. |
||||||||||||||||||||||||
With
minerals, it's not just the concentration in
the soil, it's the "availability"
and that depends in part upon pH
SOURCE: Graph modified from: http://hcs.osu.edu/hcs/TMI/HORT234/Soils/sld028.htm
Resource reading on iron deficiency
Joe Nunez
UCCE Farm Advisor, Kern County
Vegetable Crops - Plant Pathology
October 10, 2000
IRON DEFICIENCY IN VEGETABLES
"Because iron is normally found in abundant quantities in most soils, low iron
content in the soil is rarely the cause of iron deficiency in most plants. Low iron
availability or mobility due to environmental and soil conditions are most often
the cause of iron deficiency problems in plants grown in arid regions. Iron salts
that have low solubility such as iron oxides, carbonates, phosphates, hydroxides
and some forms of insoluble chelates are created in certain soil types that make
iron not readily available to plants."
continued: http://www.co.kern.ca.us/farm/NUNPUB05.HTM
See also, discussion of siderophores on pp. 364-366
Presence
of beneficial microorganisms
Inoculation
If you plant soybeans in a field that has never had soybeans in it, the microbial
flora may or may not support nitrogen fixation. There is a specific correspondence
between the species of legume and the species of rhizobium that works with it. So
what do you do if you want to plant soybeans in a new field, or a field that hasn't
been put to that purpose for a few years? You inoculate the
plants so you can get root nodules. On sandy soils you may need to inoculate every
year, because the level of survival of the rhizobium may be low.
You can inoculate the seed with the rhizobium before planting, or inoculate the soil directly. In either case, the result should be 5-7 root nodules per primary root. If the inoculation doesn't work, chemical fertilizer will be needed to replace the nitrogen that would have been contributed from the N-fixing bacteria. Why might it not work? Treatment of the seeds with fungicides of certain types may reduce the survival of the bacteria, and the rhizobium may not do well if the soil pH is too low. You may have to "lime" the soil to increase the pH above 6, so that the mineral molybdenum is "available".
Read more here: http://www.ianr.unl.edu/pubs/fieldcrops/g737.htm
Developing
the relationship
Plants secrete isoflavonoids that signal the bacteria to their presence. Bacteria
also have a set of responses that develop as they move to infect the root thread.
If the soil's too cold, it is difficult to get this started. Fortunately, you can trick the bacteria into responding to an artificial signal, for example "SoyaSignal" is a product for getting early nodulation while the soil is cold.
Introduction to nitrogen fixation - resource reading
Inoculation of legumes - resource reading
The nitrogenase enzyme
Fig 14.1 - model for the iron-molybdenum cofactor bound to a nitrogen molecule
Fig 14.2 - Assay for nitrogenase using acetylene as a test molecule
Isolating the nif genes
Fig 14.3 - Cloning nif genes by complementation
Fig 14.4 - Arrangement of genes in K. pneumoniae nif cluster
Could the nitrogen fixation pathways be transferred to plants by genetic engineering?
The
hydrogen gas problem
Reduction of H+ to hydrogen gas by nitrogenase - this is costly to the microorganism energetically
Possible solution - Bradyrhizobium japonicum hydrogenase enzyme.
Table 14.4 - Percentages of native strains with functional hydrogen uptake systems (Hup+)
Isolation of hydrogenase genes by genetic complementation
Described in third paragraph, p. 358. Broad host-range cosmid vector used to complement Hup- B. japonicum.
Results - Table 14.5. effect on plant size, leaf area.
Nodulation
Miloslav Kaláb, Ph.D. of University of Lund, provides pictures of nodules and
bacteria:
http://distans.livstek.lth.se:2080/rootnodules.htm
 |
| Nodules full of nitrogen-fixing bacteria on the roots of a soya plant Picture Source: http://distans.livstek.lth.se:2080/rootnodules.htm |
P. 362: Events in nodulation: NodD gene product, constitutively expressed, recognizes
and binds to flavonoid, and is activated. This is a determinant of host specificity.
The flavonoid-NodD complex binds to a "nod box" and turns on the genes.
Nod gene isolation
The genetic basis of "competitiveness" for nodule formation
- Cosmid library generation from R. meliloti (Nod+).
- Infect E. coli to carry library
- Conjugate E. coli with Nod- strain of R. meliloti
- Inoculate alfalfa
Table 14.6 Nodulation gene products
Competition among nodulating organisms
Why do the indigenous bacteria have an advantage? Nobody knows!
- Identification of nodulation (nod) genes of Rhizobium by complementation (pp. 359-361, Fig 14.6)
- Follow-up probing to identify genes in adjacent regions
| Example
of a patent on file: http://www.nal.usda.gov/bic/Biotech_Patents/1995patents/05427785.html BACKGROUND OF THE INVENTION The identification or production of microbial strains with enhanced biological activity, e.g., strains with enhanced nitrogen fixation capability, or enhanced pesticidal activity, is an important goal in agricultural biotechnology. In many agricultural applications an improved microbial strain is added to the soil and thus must compete with native free-living microbes for growth and nutrients. Even though a microbe possesses a useful biological activity, failure to compete with native organisms will most often render it ineffective in the field. For example, displacement of established populations of Bradyrhizobium japonicum by inoculation with strains that are efficient nitrogen fixers has proven difficult (Triplett (1990) Molec. Plant Microb. Interactions 3:199-206). Efforts to improve the competitiveness of specific microorganisms in agricultural settings have included searches for naturally occurring strains which possess a competitive advantage, Tn5 mutagenesis-selection experiments, and experiments designed to exploit the effects of various naturally occurring indigenous plasmids. SUMMARY OF THE INVENTION In general the invention features a bacterial inoculum including a rhizospheric bacterium with increased dicarboxylic acid membrane permease activity. In preferred embodiments the rhizospheric bacterium is transformed with a DNA sequence which increases the dicarboxylic acid membrane permease (DMP) activity of the bacterium, e.g., a sequence which encodes dicarboxylic acid membrane permease, or any or all of: a sequence which encodes the dctB gene product, a sequence which encodes the dctD gene product, or a sequence which encodes the dctA gene product. In preferred embodiments the bacterium of the inoculum is transformed with a second DNA sequence, the second sequence conferring a desirable property on the bacterium e.g., a sequence which increases the capability of the bacterium to fix nitrogen e.g., a sequence encoding a nifA gene product. In preferred embodiments of the inoculum the rhizospheric bacterium is capable of colonizing the rhizosphere of a soybean plant; the rhizospheric bacterium is in the genus Bradyrhizobium, and is preferably Bradyrhizobium japonicum; the rhizospheric bacterium is capable of colonizing the rhizosphere of an alfalfa plant; the rhizospheric bacterium is in the genus Rhizobium, and is preferably Rhizobium meliloti; the rhizospheric bacterium is capable of colonizing the rhizosphere of a clover plant; and the rhizospheric bacterium is Rhizobium trifolii. |
Biocontrol of pathogens
Siderophores
- An historical problem - oxygenation of Earth, generation of unavailable ferric iron Fe(III) with a solubility of 10 -18 molar at pH 7.4
- A solution - secretion of iron-binding molecules (siderophores) that can transport iron back to microbial cell.
The good news for plants...besides the fact that they can grow at a lower concentration of iron than microorganisms... is that:
- Plant growth-promoting bacteria may prevent proliferation of fungal phytopathogens by secretion of siderophores that out-compete the fungi for the iron.
- Example of siderphore (Fig. 14.8)
Cloning of genes for siderophore production in Pseudomonas by complementation (pp. 365-6)
- Generation of siderophore - nonproducing strains by mutagenesis
- criteria are lack of UV fluorescence and inability to grow in bipyridyl (i.e. low iron conditions)
- Construction of clone bank from P. putida DNA in broad host-range plasmid
- Conjugation with clone bank host
- Selection (bipyridyl) and screening (fluorescence) for siderophore production
Antibiotic expression by biocontrol pseudomonads
Antibiotic-secreting, plant growth-promoting bacteria
http://apc-online.com/twa/agriculture2.shtml
Tomorrow's World, The Australian Initiative
Environmentally friendly non-chemical pesticide
Increasingly in the nineties, research scientists are working to find environmentally friendly solutions to pest control.
Researchers at the Waite Agricultural Research Institute, University of Adelaide in conjunction with Bio-Care Technology Pty Ltd have developed a new, non-chemical biological control agent. Invented by Professor Allen Kerr in 1988, the active ingredient of NOGALL is a bacterium called Agrobacterium radiobacter, strain KI026, which is incorporated into a moist peat pure culture. It is used as a biocontrol treatment against crown gall disease in stone fruits, nut trees and roses.
NOGALL is applied as an aqueous suspension to seeds, seedlings and cuttings before planting, and works by protecting wound sites from infection. For effective action it has to be applied within two hours of damage caused by taking cuttings, repotting, lifting and planting techniques.
The NOGALL bacteria belong to a non-disease causing species of the crown gall bacteria group and act as a biological control agent primarily due to the production of a neutral antibiotic called agrocin 84. The key to the development of the strain KI026 was the removal of a small piece of DNA from the bacterium. This prevents the transfer of the agrocin 84 genes to other soil bacteria, and hence prevents any immunity build-up against the protective nature of strain KI026. Containing strain KI026, NOGALL was the first live genetically-engineered micro-organism made available to the public.
Crown gall disease causes hundreds of millions of dollars worth of damage to crops worldwide. With the exceptional protective benefits of NOGALL, horticulturalists around the world can expect to obtain greatly improved crop returns and significant savings for consumers.How is transfer of a resistance plasmid to the pathogenic species of Agrobacterium prevented?
- See Fig. 14.9 (p. 368)
Prevention of Ice Nucleation
An item from the California Rare Fruit Growers Journal
"The Christmas weekend freeze of 1987 had a sobering effect on my enthusiasm for growing many of the conditionally adapted exotic fruit trees here in Southern California."
"Ice-minus" mutant strains of Pseudomonas
Resource: slides for
chapter 14
Microbial
Insecticides
FROM THE PATENT FILE
http://www.nalusda.gov/bic/BTTOX/BT-patents/05279962.html
B. thuringiensis has been used for many years for the production of insecticides, but although mutants of B. thuringiensis with increased delta-endotoxins yield would be advantageous, no such mutants have previously been described. Mutants producing higher yields of delta-endotoxins would give a more efficient and economical production of B. thuringiensis toxins and a possibility for manufacture of B. thuringiensis products with increased potency at equal cost. This in turn would be an advantage for the user as reduced volumes of pesticide formulation have to be stored and handled for a given acreage. In addition, the users will have less container material to dispose of, thereby reducing the impact on the environment.
Improvements of the production of delta endotoxin by Bacillus thuringiensi s subsp. tenebrionis through mutation have not previously been reported.
One problem associated with the use of especially B. thuringiensis subspecies tenebrionis in controlling beetle larvae has been the relatively low potency or strength of the preparations requiring the application of relatively large amounts of preparation to the areas to be treated, such as 5 to 10 liter/ha compared to 1 to 2 liter/ha of most other B. thuringiensis products and most other insecticides.Consequently a recognized need for products of improved strength exists.
One way to overcome this problem would be to concentrate the preparations. However, this would add considerably to the production cost in comparison to the savings obtained in storage and transportation.
A much more elegant solution would be to create mutants of existing B. thuringiensis strains capable of producing substantially larger amounts of delta endotoxins per cell.
SUMMARY OF THE INVENTION
The present invention consequently in one aspect relates to variant or mutant Bacillus thuringiensis strains capable of producing substantially larger amounts of toxins than their parent strain.
In another aspect the invention relates to such high producing variants or mutants of B. thuringiensis strains belonging to the subspecies tenebrionis.Mode of action of parasporal crystals (pp. 378-381)
- ingestion of B. thuringiensis is required
- limitation of killing to insects in specific developmental stage
- pro-toxin activation in gut (alkaline pH, specific digestive proteases)
- creation of ion channel (concomitant expenditure of ATP)
Forms of resistance to B. thuringiensis
- alteration in midgut membrane protein that is receptor for toxin
Isolation of toxin genes from B. thuringiensis plasmids (Fig 15.3, p. 383)
- fractionation of plasmids by CsCl gradient centrifugation (in presence of ethidium bromide)
- fractionation by sucrose gradient centrifugation
- subcloning into an E. coli library
- use of rabbit anti-protoxin antibodies to detect colonies expressing protein (125I-protein A mediated)
Suggested applications
Constitutive expression of protoxin (i.e. not just during sporulation) - see Fig 15.4, p. 385
Modification of host toxicity range of cry gene products
The natural order of things...
Table 15.1 (abridged) p. 379
strain or subspecies Toxin class Target Pictures from the Tree of Life berliner, kurstaki KTO, entomocidus, aizawai 7.29 CryI Lepidoptera
aizawai IC 1, kurstaki HD-1 CryII Lepidoptera, Diptera
tenebrionis (san diego) CryIII Coleoptera
israelensis CryIV Diptera
Toxicity of transformed strains of aizawai, israelensis, and tenebrionis (Fig 15.2, p. 387)
Toxicity of hybrid cry gene fusion products (CryIC and CryIE, Fig. 15.5, p. 388)
- Separation of toxic domain III from domains fostering resistance
Alteration of location of microbial insecticide
- Synechocystis vs. B. thuringiensis for control of mosquitoes (p. 388)
- Pseudomonas vs. B. thuringiensis for control of insects attacking roots (p. 389)
- Method of cloning by Tn5 tagging of P. fluorescens and homologous recombination
Expression of insect toxins in baculoviruses
Table 15.4 (p. 392) Gene Effect on insect Diuretic hormone Reduced hemolymph volume Juvenile hormone esterase Feeding cessation B. thuingiensis toxin Feeding cessation Scorpion toxin Paralysis Mite toxin Paralysis Wasp toxin Low weight gain
See the slides for chapter 15
Taking a peek at Chapter 18Introduction of protoxin genes into plants, for direct expression
 |
Reading Assignment Chapter 16 |