Diagnosis of Plant Diseases
Authors: Deepak V Pawar1, Mahesh Mahajan1, Rakesh Kumar Prajapat1, Kishor U Tribhuvan1
1ICAR-NRCPB, I.A.R.I, New Delhi-12


Agricultural crops are threatened by various diseases and pests which can damage crops, lower harvest quality or even destroy entire harvests. Almost half of the world's total harvest is destroyed by diseases and pests annually. Moreover, farmers often deal with several pests or diseases and new pesticide-resistant pathogens. If diagnosed early and correctly, treatments can be developed against these pathogens and could minimize losses. The common method of diagnosis is visual examination, which is often only possible after crops have been damaged. Hence, farmers should identify an infection before it becomes visible. Pathogen infection in plants causes a complex immune response, producing proteins involved in plant defense. Pathogens also produce proteins and toxins to help their infection before disease symptoms appear. These molecules are crucial in the development of plant diagnostic kits. Advances in molecular biology, plant pathology, and biotechnology have made the diagnostic kits possible. These detect diseases early, either by sensing the presence of the pathogen's DNA or the proteins produced by either the pathogen or the plant during infection. These require minimal processing time and are more accurate in identifying pathogens. Although some require laboratory equipment and training, other procedures can be done on-site by a person with no training. Diagnostic kits have been designed to detect diseases in crops such as rice, potatoes, papaya, tomatoes, and banana. Similar kits are also used for identifying genetically modified organisms (GMOs) in shipments of conventional crops.

DNA-based diagnostic kits

DNA diagnostic kits are based on the ability of single stranded nucleic acids to bind to other single stranded nucleic acids with a complementary sequence. The tool used in DNA diagnostic kits is the Polymerase Chain Reaction (PCR). There are 3 steps involved in PCR:
1. The DNA is unwound, and its strands are separated by high temperatures
2. As the temperature is lowered, short, single-stranded DNA sequences called primers are
free to bind to the DNA strands at regions of homology
3. This allows the (Taq) polymerase enzyme to make a new copy of the molecule.

This cycle is repeated 30-40 times, yielding millions of identical copies of the segment. The primers in PCR diagnostic kits are very specific for the genes of a pathogen, and DNA amplification will occur only in diseased plants. Several PCR-based methods have successfully been adapted for plant pathogen detection such as the real-time PCR (RT PCR). It follows the principle of PCR but quantifies amplified DNA using fluorescent dyes as it accumulates after each cycle. It offers several advantages over normal PCR, including reduced risk of sample contamination, real time data and simultaneous testing for multiple pathogens. DNA microarrays are also of great use for simultaneous pathogen detection since plants are often infected with several pathogens at once, sometimes causing 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. PCR-based diagnostics are sensitive enough to detect small amounts of DNA. PCR can also help farmers detect pathogens with long periods between infection and symptom development. Moreover, it can quantify pathogen biomass. PCR kits have been developed for black Sigatoka disease in bananas, Phytophthora infestations in potatoes, and Fusarium infection in cotton. However, PCR-based detection is expensive and requires expensive equipment.

Protein-based diagnostic kits


The first step in a defense response reaction is the recognition of an invader by the host's immune system. This recognition is due to the ability of host proteins, called antibodies, to recognize and bind proteins that are unique to a pathogen, called antigens, and trigger an immune reaction. Protein-based diagnostic kits for plant diseases contain a primary antibody that can recognize a protein from either the pathogen or the infected plant. It also contains a secondary antibody which is joined to an enzyme. This enzyme will catalyze a chemical reaction that causes a color change when the primary antibody is bound to an antigen signaling the presence of the pathogen.

The enzyme-linked immunosorbent assay (ELISA) method uses this detection system, and is the basis of some protein-based diagnostic kits. ELISA kits are very easy to use, takes only a few minutes to do, and does not require special laboratory equipment or training. There are already numerous ELISA test kits available on the market, including diagnostic kits for root crops, ornamentals, fruits, grains, and vegetables. One of the first ELISA kits for diagnosing plant diseases was from the International Potato Center (CIP). It can detect the presence of all races, biovars, and serotypes of Ralstonia solanacearum, the causal agent of bacterial wilt or brown rot in potato. They also developed a kit that detects the presence of sweet potato viruses.


Conclusion

With even more advances in molecular biology and immunology, scientists and farmers alike can improve plant disease diagnosis. Development of better diagnostic kits for important crops is already underway. Diagnostic kits may be expensive but gains from it are definitely worth it.


About Author / Additional Info:
I am PhD research scholar, pursuing PhD at IARI, New Delhi in the discipline of Molecular Biology and Biotechnology. I am working on blast disease resistance in O. sativa