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Biotechnology in Plant Disease Control

P. Balasubramanian

Now that it has become routine to transfer genes from one organism to another, it is possible to introduce genes conferring disease resistance into crop plants. Such gene transfers could be accomplished by two methods, direct methods and vector-mediated methods. Gene gun or Biolistic method and Agrobacterium-mediated method are the best examples of the direct method and vector-mediated method respectively.
Transgenic Plant Disease Management

Disease resistance genes could be sourced from plant pathogens themselves, as was possible with coat protein-mediated plant viral resistance and with toxin-inactivating protein-mediated bacterial resistance. Host plants also contribute an enormous number of disease resistance genes such as those encoding pathogenesis-related (PR) proteins, which have been used against fungal diseases.
Candidate genes against viral pathogens
One of the most successful examples, as of date, of the use of transgenic resistance against plant diseases is that was accomplished in the management of papaya ring spot virus (PRSV) in Hawaii. Traditional breeding in bringing about resistance against this disease was of no avail, as crossability barriers were a big problem. Under these circumstances, coat-protein-mediated resistance using coat protein gene sourced from a Hawaiian strain of PRSV was attempted. One transgenic line was found to be completely resistant to PRSV.
Transgenic resistance against banana bunchytop resistance using a BBTV replicase gene is under study. However, it might take some more time to be successful in this attempt, as we have yet to accomplish much in the routine generation of transgenic banana lines of local importance. Once generation of transgenic banana has become routine, it will be easier for us to deliver human vaccines (cholera toxin vaccine and hepatitis B surface antigen) via transgenic banana fruits, as banana fruit forms an excellent delivery material.
Recently, a gene silencing mechanism has been put to productive use in containing rice yellow mottle virus.  An open reading frame of the virus itself is expressed in rice in order to stop the viral spread in an effective manner. Similar attempts also have been made in containing multiple viral infections (tomato spotted wilt virus and turnip mosaic virus) in plants.
Candidate genes against bacterial pathogens
Xa21, a wide-spectrum bacterial blight resistance gene sourced from an African rice, Oryza longistaminata was backcrossed into a cultivated variety by scientists  of the International Rice Research Institute, the Philippines (IRRI). The resistance gene was cloned using molecular means by Pam Ronald of University of California and distributed to labs all over the world, so that the gene could be put into rice cultivars of local importance.
Wild fire disease of tobacco caused by Psuedomonas syringae pv. tabaci is a serious disease.  A phytotoxin secreted by the pathogen drastically modifies the amino acid metabolism of the plant with the eventual accumulation of ammonia in tobacco leaves, which causes extensive blighting. Interestingly, the pathogen that synthesises the phytotoxin remains unaffected by the toxin. This formed the basis for a search of the candidate gene from the pathogen itself.  A toxin-inactivating gene, which was named ‘ttr’ was successfully isolated from the pathogen and the same was cloned into tobacco cultivars, which showed excellent wildfire resistance.
Candidate genes for fungal resistance
PR protein genes appear to be a very potential source for candidate genes for fungal resistance. These proteins may play a direct role in defense by attacking and degrading pathogen cell wall components.  Typical candidate genes are that encoding chitinases and ß-1,3 glucanases. Increasing expression of individual and multiple PR-proteins in various crops have demonstrated some success in enhancing disease resistance in particular pathogens (e.g., in rice against Rhizoctonia solani, the sheath blight pathogen). A recent research shows a chitinase gene from an anti-fungal biocontrol fungus species (Trichoderma viride) confers transgenic resistance against the rice sheath blight pathogen. A rice PR-5 protein gene in wheat delays onset of symptoms caused by the wheat scab pathogen.
Biocontrol of Plant Pathogens

The increased reflection on environmental concern over pesticide use has been instrumental in a large upsurge of biological disease control.  Among the various antagonists used for the management of plant diseases, Trichoderma and Pseudomonas play a vital role.
Trichoderma, a successful bio-control agent against plant diseases
Among the various isolates of Trichoderma, T. viride, T. harzianum, T .virens and T. hamatum are used against the management of various diseases of crop plants especially with dreaded soil-borne pathogens. It has many advantages as a bio-control agent owing to its high rhizosphere competence, ability to synthesize polysaccharide–degrading enzymes, amenability for mass multiplication, broad spectrum of action against various pathogens and above all its environmental friendliness.
Fluorescent pseudomonads in induced systemic resistance (ISR) against plant diseases
Fluorescent pseudomonads suppress the pathogens either directly through the production of various secondary metabolites or indirectly by inducing plant-mediated defense reactions. The crucial factor in the success of biological control by fluorescent pseudomonads is their ability to colonize the rhizosphere and their persistence throughout the growing season. Fluorescent pseudomonads are root colonizers because they occur in the natural habitat of rhizosphere and thus when they are reintroduced to roots through seed or seed-piece inoculation, they colonize root surface profusely. Fluorescent pseudomonads suppress the pathogens by antibiosis through the production of various antibiotic substances such as 2,4-diacetyl phloroglucinol, phenazine-1-carboxylic acid, oomycin A, oxychlororaphine, pyoluteorin, pyrrolnitrin and pyocyanine. Siderophores are extracellular, low molecular weight substances which selectively complex iron with high affinity. Fluorescent pseudomonads produce siderophores such as pseudobactin and pyoverdine which chelate the iron available in the soil and make it unavailable to pathogen thus the pathogen dies for want of iron.
In rice, seed treatment followed by root dipping and foliar spray with Pseudomonas fluorescens showed a higher induction of ISR against sheath blight pathogen, R. solani
Molecular Diagnostics of Plant Diseases

The first and most important step in managing a plant disease is to correctly identify it. Although some diseases can be diagnosed quickly by visual examination, others require laboratory testing for diagnosis. These laboratory procedures may take days or even weeks to complete and are, in some cases, relatively insensitive. Delays are frustrating when a quick diagnosis is needed so that appropriate disease control measures may be taken to prevent plant injury, especially when high value cash crops are at stake. Fortunately, as the result of advances in biotechnology, new products and techniques are becoming available that will complement or replace time consuming laboratory procedures.
ELISA Diagnostic Kits
A number of disease detection kits have been developed for use at the site where a disease is suspected. These kits, which in most cases do not require laboratory equipment, are especially useful to growers. Some tests only take five minutes to perform. The diagnostic kits are based on a method that uses proteins called antibodies to detect disease causing organisms of plants (plant pathogens). The technique used is called ELISA (enzyme-linked immunosorbent assay). This assay is based on the ability of an antibody to recognize and bind to a specific antigen, a substance associated with a plant pathogen. The antibodies used in the diagnostic kits are highly purified proteins produced by injecting a warm-blooded animal (like a rabbit) with an antigen associated with one particular plant disease. The animal reacts to the antigen and produces antibodies. The antibodies produced recognize and react only with the proteins associated with the causal agent of that plant disease. Color changes on the unit’s surface indicate a positive (disease present) reaction. Examples of diseases ELISA kits can detect include bacterial canker of tomato and soybean root rot.
A new technology, PCR (polymerase chain reaction) has great potential for raising the sensitivity of various assays that use nucleic acid probes. PCR is used to produce enormous numbers of copies of a specified nucleic acid sequence. This technique can allow the detection of very small amounts of a pathogen in a sample by amplifying the pathogen sequences to a detectable level. PCR is especially useful in plant quarantine point of view owing to its fastness.

P. Balasubramanian
Professor and Head, Department of Biotechnology,
Centre for Plant Molecular Biology, Tamil Nadu Agricultural University
Coimbatore 641 003, India, balasubrap@hotmail.com