Table 2.
Advantages and disadvantages of different detection and diagnostic approaches for R. solani associated with legume crops.
| Diagnostic methods | Assays/Platforms | Advantages | Disadvantages |
|---|---|---|---|
| Traditional approaches | Visual examination | Symptom-based | Symptoms common to many pathogens |
| Cheaper | Not suitable for latent infection | ||
| Incubation methods | Good for high-incidence fungi | ||
| Providing information about the viability | |||
| Cheaper | Fungal reproductive structures are not always produced on agar media Ambiguous nature of anastomosis grouping of isolates |
||
| Simplicity of application | Time-consuming | ||
| Require mycological skills | |||
| Low sensitivity | |||
| Not always reliable | |||
| Low specificity | |||
| Microscopy | Right-angle branching of septate hyphae | Cannot differentiate AGs or AGs subgroups of R. solani | |
| Biochemical approaches | Fatty acid profiling, and | Helpful in examining genetic diversity among AGs | The lack of specificity of the antibody |
| Pectin enzyme analysis | Commonly not used for direct soil or plant material testing | ||
| Isozyme polymorphism | Also, require skilled persons | ||
| Serological methods | Do not require pure isolation of the pathogen | Cannot distinguish AGs subgroups of R. solani | |
| Applicable to R. solani, which is a necrotrophic pathogen | |||
| Lack of species-specific antibodies | |||
| cannot distinguish between pathogenic and non-pathogenic | |||
| Detect non-viable pathogens, which can result in erroneous interpretations | |||
| Matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) | Analysis of non-volatile high-molecular compounds (peptides, proteins, carbohydrates, oligonucleotides, synthetic polymers, organic complex compounds, etc.) of R. solani |
Required pure culture of R. solani | |
| Polymerase chain reaction (PCR)-based approaches | Conventional PCR | Rapidity, specificity, sensitivity, and easy interpretation | Compounds inhibit DNA amplification, resulting in false negatives |
| Distinguish between closely related organisms | Gives only qualitative data | ||
| The presence of low levels of inoculum can be a problem and may result in a false negative | |||
| Cannot distinguish between viable and non-viable inoculum of R. solani | |||
| BIO-PCR | Detect fungus at very low levels | ||
| Highly sensitive PCR technique | |||
| Elimination of PCR inhibitors | |||
| Detection of viable cells | |||
| Avoiding false positives | More expensive than conventional PCR, primarily if selective media are used | ||
| DNA–DNA hybridization assay | DNA relatedness in AGs |
Requires the entire genome of the species | |
| Time-consuming pairwise comparison | |||
| Nested PCR | Detection of a target DNA at several-fold lower levels | More labor intensive | |
| More costly | |||
| More prone to contamination | |||
| Real-time PCR | Allows quantification of specific DNA targets | ||
| Reduces the risk of false positives due to cross-contamination of the reaction mixtures | |||
| Less time-consuming | |||
| High sensitivity | Issues with sensitivity, repeatability, and specificity | ||
| Use multiple primers to reduce costs and labor | Need appropriate target DNA fragments for the design of the primers and probes is problematic | ||
| SCAR Approach | Amplify members from the same genus | ||
| High specificity using soil or infected plant parts | Need for sequence data to design the PCR primers | ||
| Quick and easy to use | Require effort and expense in primers designs | ||
| They have high reproducibility and are locus-specific | |||
| Fingerprinting Techniques | Amplify random tandem repeats on genomic DNA | Necessitate pure fungal cultures and are not ideal for directly exploring plant material, soil, or growing media | |
| Detect species-specific patterns | |||
| Phylogenetic structure of different microbial species | |||
| understanding of population structure | |||
| Cross-Hybridization using UP-PCR | A single UP-primer is used to determine the sequence similarity (homology) of unknown Rhizoctonia strains | Temperature of hybridization and salt concentration | |
| Concentration of the denaturant in the buffer | |||
| Length and nature of the probe sequence. | |||
| Transcriptomic approaches | Closely related strains of R. solani with distinct characteristics may be gleaned through comparative sequencing analysis | Need intensive work with massive sequencing data because of the R. solani multinucleate nature | |
| Genomic approaches | Draft genome sequence | Extensive host range and virulence | |
| Loop-mediated isothermal amplification (LAMP) | Simple | ||
| Cost-effective | |||
| A rapid method for specific detection of genomic DNA | |||
| All of the reactions can be carried out under isothermal conditions | |||
| It does not require expensive equipment | |||
| Fewer preparation steps | Heavy reliance on indirect detection methods like turbidity and non-specific dyes, often leads to the detection of false positive results. | ||
| Highly specific | |||
| The amplification efficiency of LAMP is exceptionally high | |||
| Next-generation sequencing | Life Sciences 454 sequencing | Analysis of RNAs | |
| Rapid identification | Computational resources required for the assembly, annotation, and analysis of sequencing data | ||
| Mycobiome can be studied | Lower per read accuracy | ||
| Taking advantage of PCR emulsion | |||
| A highly efficient in vitro DNA amplification method | |||
| Can produce 80–120 Mb of sequence in 200- to 300-bp reads in a 4 h run | |||
| AB/SOLiD technology | Sequencing by oligonucleotide ligation and detection (SOLiD). | Computational resources required for the assembly, annotation, and analysis of sequencing data | |
| Employs few inputs and is based on chemistry involving the ligation of di-base labeled probes. | Lower per read accuracy | ||
| The typical read length for a SOLiD run is 25–35 bp, and the total amount of sequencing data generated is 3–4 Gb for 5 days | |||
| Illumina/Solexa sequencing | Similar to the Sanger-based methods | Computational resources required for the assembly, annotation, and analysis of sequencing data | |
| Solexa terminators are reversible, permitting the continuation of polymerization following fluorophore detection and deactivation | Lower per read accuracy | ||
| Solid-phase amplification involves the immobilization of sheared DNA fragments on a solid surface (flow-cell channel) | |||
| The average run size is 40–50 Mb (read duration is 50–300 bp) |