Different Methods for Identification of Bacterial Plant Diseases
Authors: Dipti Raghunath Dhumale, Prashant Raghunath Shingote and Yashoda Bhausaheb Etther

Precise and perfect identification bacterial and other pathogen is necessary for correct disease diagnosis. This also helps in treatment of disorder and to find disease outbreaks associated with infections. Variety of other important applications of bacterial identification including: plant/seed quarantines, evolutionary studies, ecological studies, diversity and pathogenicity tests, bio-terrorism, illegal investigations etc. Bacterial identification by conventional technique is depends on symptomatic categorization of the pathogen, pathogenicity and hypersensitivity tests, Gram staining, culture characters, colony appearance, media utilization tests and biochemical methods. On the other hand, above described technique face some major lacunas like: first requirement is to have pure and In vitro grown cultures of causal organisms and secondly, some biochemical characters can be same for diverse group of organisms. As the discoveries in molecular techniques it has proved that lacunas of traditional methods for the detection and characterization can be overcome. Numerous non-culture based rapid and reliable methods have emerged about in the past two decades. PCR, Real time PCR and microarrays are the frequently used molecular techniques. These are highly responsive, rapid and allow quantitation of bacteria at a species level. Hybridization based microarray is technique relies on hybridization of species-specific oligonucleotides (Probe) with amplicons of the bacterial DNA sequences. These oligoneucleotide are tagged with a dye of different color which fluoresces at hybridization only.

Bacterial Identification through PCR:

Application of PCR technology with different DNA based oligonecleotides primers (Universal and specific) can detect bacteria directly from infected tissue samples or from little amounts of pure bacterial cultures (Shingote et al. 2013a, Shingote et al. 2013b). The PCR based methods are precise and also reduces the time for analysis of large number of samples. Oligonecleotide primers, which are designed from sequences of bacterial genome either may specific to particular gene (pig, hrp), repeat elements (XRIC, ERIC) or form universal genes(16S/23SrRNA, 16S-23S ITS) . Sequences of amplicons generated from these primers help in bacterial diagnosis at level of strain, species or genus. Moreover these techniques have been also illustrated for diversity analysis of several bacterial isolates as well. PCR allows researchers to study very small amounts of DNA without resorting to laborious cloning procedures.

Now a day's an advanced method in this amplification based diagnosis a Real Time PCR (RT-PCR). By using this DNA based assay, it is possible to detect bacterial strains form minute samples, improved sensitivity as well shortened for identification are the characteristics of this techniques. Real-time PCR is a robust tool for distinguishing particular sequences from a mixed bacterial culture as well mixture of DNA and hence it is helpful for detecting the existence and number of unique sequences within a sample tested. Real-time PCR helps in rapid detection of low amounts of bacterial DNA facilitate in advance disease management strategies in field level.

Bacterial Identification through DNA sequencing:

PCR amplicons of the universal primers (16S rRNA gene) using DNA of unknown bacterial strain can be sequenced for proper taxonomic placements. After determining the DNA sequence of the 16S rRNA genes; by comparing the generated sequence to a computerized database of already known sequences, one can determine a "molecular taxonomic classification" of the unknown bacteria. DNA Analysis allows for samples to be compared directly to each other, and data can be interpreted in a number of figures and designs. These include the familiar Neighbor Joining trees and Sequence Alignment tools which may be useful for evolutionary and comparative studies.

Bacterial Identification through Microarray:

A microarray has a potential of identification and speciation of bacterial strains in side by side. The technique is multipurpose and makes it possible to identify and distinguish multiple bacterial test samples on a kit. Since the fast classification of the bacteria in field samples is an imperative task for efficient control management the DNA microarray as hybridization based approach promising. This diagnosis of suspect is by using species-specific oligonucleotide probes designed for particular section of targeted genes. These probes are tagged with a fluorescent dye of different color, which gives signals only at hybridization. The presence of fluoresce confirms the existence of particular gene which leads to revealing the suspected causal bacteria in test sample.

Conclusion:

Bacterial pathogen is needs to be diagnosed whether from animal or plant. This assists to the cake their outbreaks. Other applications of identification are helpful in quarantine, diversity, bio-terrorism and investigations areas. Conventional techniques are laborious, painstaking and time intensive also unable to organisms appropriately and reproducibly. Evolution of molecular techniques leads to conquering of traditional methods. Numerous rapid and reliable methods have been emerged like: PCR, DNA sequencing, Real time PCR, microarrays and GC-FAME etc. Most of the technologies were preference fully utilized for medical applications but by the development of need and technology it is also found beneficial in agriculturally important bacterial disease identification.

References:

1. Shingote PR, Moharil MP, Dhumale DR, Jadhav PV, Satpute NS, Dudhare MS (2013) Screening of vip1/vip2 binary toxin gene and its isolation and cloning from local Bacillus thuringiensis isolates. Sci Asia 39:620-624

2. Shingote PR, Moharil MP, Dhumale DR, Deshmukh AG, Jadhav PV, Satpute NS, Dudhare MS (2013) Screening Distribution of vip Genes, Protein Profiling and Determination of Entomopathogenic Potential of Local Isolates of Bacillus thuringiensis. Bt research 4(3): 14-20


About Author / Additional Info:
I have completed my graduation and post graduation in Agricultural biotechnology. From last 5 years I have been working on different aspects of plant biotechnology.