Dr. Raju Ghosh
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) E-Mail: r.ghosh@cgiar.org
Important agricultural crops are threatened by a wide variety of plant diseases and pests. These can damage crops, lower fruit and vegetable quality and wipe out entire harvests. About 42% of the world's total agricultural crop is destroyed yearly by diseases and pests. Farmers often must contend with more than one pest or disease and new pesticide-resistant pathogenic strains attacking the same crop.
However, crop losses can be minimized, and specific treatments can be tailored to combat specific pathogens if plant diseases are correctly diagnosed and identified early. These need-based treatments also translate to economic and environmental gains.
The traditional method of identifying plant pathogens is through visual examination. This is often possible only after major damage has already been done to the crop, so treatments will be of limited or no use. To save plants from irreparable damage by pathogens, farmers have to be able to identify an infection even before it becomes visible.
Is this possible? What happens when pathogens attack a plant? An attack by disease-causing organisms generates a complex immune response in a plant, resulting in the production of disease-specific proteins involved in plant defense and in limiting the spread of infection. Pathogens also produce proteins and toxins to facilitate their infection, before disease symptoms appear. These molecules play vital role in the development of plant diagnostic kits.
Advances in molecular biology, plant pathology, and biotechnology have made the development of such kits possible. These kits are designed to detect plant diseases early, either by identifying the presence of the pathogen in the plant (by testing for the presence of pathogen DNA) or the molecules (proteins) produced by either the pathogen or the plant during infection. These techniques require minimal processing time and are more accurate in identifying pathogens. And while some require laboratory equipment and training, other procedures can be performed on site by a person with no special training.
So far, diagnostic kits have been designed to detect diseases in crops such as chickpea, pigeonpea, rice, potatoes, papaya, tomatoe, groundnut, jute, mesta, apple and banana.
DNA-Based Diagnostic
DNA diagnostic are based on the ability of single stranded nucleic acids to bind to other single stranded nucleic acids that are complementary in sequence. The tool used in DNA diagnostic is the Polymerase Chain Reaction (PCR). There are 3 steps involved in PCR. The DNA is first unwound, and its strands separated by high temperatures. As the temperature is lowered, short, single-stranded DNA sequences called primers are free to bind to the DNA strands at regions of homology, allowing the TaqDNA polymerase enzyme to make a new copy of the molecule. This cycle of denaturation-annealing- elongation is repeated 30-40 times, yielding millions of identical copies of the segment.
The primers in PCR diagnostic are very specific for the genes of a pathogen, and amplification will occur only in diseased plants. (Figure 1)
Figure 1: PCR-based Diagnostic Methods
Several PCR-based methods have successfully been adapted for plant pathogen detection. Real-time PCR (RT-PCR) follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified, using fluorescent dyes, as it accumulates in the reaction mixture after each cycle. It offers several advantages over normal PCR, including: reduced risk of sample contamination, provision of data in real time and simultaneous testing for multiple pathogens. Real- time PCR protocols are among the most rapid species-specific detection techniques currently available.
DNA microarrays are also of great use for simultaneous pathogen detection. This is important, as plants are often infected with several pathogens, some of which may act together to cause a disease complex. Microarrays consist of pathogen- specific DNA sequences immobilized onto a solid surface. Sample DNA is amplified by PCR, labeled with fluorescent dyes, and then hybridized to the array (Figure 2).
Figure 2: DNA microarray
PCR-based diagnostics is very sensitive compared to other techniques; detection of a small amount of DNA is possible. PCR can also help farmers detect the presence of pathogens that have long latent periods between infection and symptom development. Moreover, it can quantify pathogen biomass in host tissue and environmental samples, and at the same time detect fungicide resistance. PCR- based detection, however, is expensive compared to protein-based diagnostic methods, and also requires costly equipments.
Recently, PCR kits have been developed to detect dry root rot, fusarium wilt, botrytis grey mold disease in chickpea, fusarium, phytophthora and sterility mosaic infection in pigeonpea.
Protein-Based Diagnostic
The first step in a defense response reaction is the recognition of an invader by a host's immune system. The recognition is due to the ability of specific host proteins, called antibodies, to recognize and bind proteins that are unique to a pathogen (antigens) and to trigger an immune reaction (Figure 3).
Figure 3: Antibody-Antigen Interaction
Protein-based diagnostic for plant diseases contain an antibody (the primary antibody) that can either recognize a protein from either the pathogen or the diseased plant. Because the antibody-antigen complex cannot be seen by the naked eye, diagnostic also contain a secondary antibody, which is joined to an enzyme. This enzyme will catalyse a chemical reaction that will result in a colour change only when the primary antibody is bound to the antigen. Therefore, if a colour change occurs in the reaction mixture, then the plant pathogen is present, (Figure 3).
The enzyme-linked immunosorbent assay (ELISA) method makes use of this detection system, and forms the basis of some protein-based diagnostic. ELISA kits are very easy to use because test takes only a few minutes to perform. There are already numerous ELISA test kits available on the market. ELISA techniques can detect potato viruses, groundnut bud necrosis virus, pigeonpea sterility mosaic virus, tomato mosaic virus, papaya ringspot virus, and rice tungro virus.
Conclusion
With even more advances in molecular biology and immunology, scientists and farmers alike will be able to improve plant disease diagnosis. Efforts are already underway to produce better diagnostic kits to detect pathogens in crops important to developing countries. For instance, the Department of Biotechnology of India's Ministry of Science and Technology is established several referral centre and accredited test laboratories to develop diagnostic kits to detect viruses in fruits, ornamentals, spices, and plantation crops.
Diagnostic kits are an investment: they may be expensive, but the costs can be offset by gains, such as reduced crop losses and more environment-friendly crop-management practices. Their development should be made a priority by both the public and private sectors in developing countries.