Jameel Akhtar, Raj Kiran, Pardeep Kumar, Meena Shekhar and SC Dubey
The Division of Plant Quarantine at ICAR-NBPGR has developed procedures for systematic and stepwise processing for detection of pathogens associated with exotic germplasm (Figure 1). Seed-borne pathogens may result in poor quality seed, loss in germination, development of epiphytotics, distribution of new strains or physiological races of pathogens along with the seeds and planting material to new geographical areas. Therefore, critical examinations with specialized testing methods are conducted to ensure pathogens-free import of seeds and other planting materials. The methods used for quarantine testing are:
(i) Visual examination (ii) Washing test
(iii) Seed soaking method
(iv) Incubation methods (Blotter/ Agar plate) (v) Molecular detection
Visual examination of seeds: Preliminary examination with naked eye or with the help of a magnifier helps detecting presence of abnormalities such as discoloration, deformation shriveling, pigmentation, malformation of seed with fungal growth like mycelial mats or fructifications like chlamydospores, acervuli, pycnidia, perithecia and other impurities associated with a seed lot such as sclerotia, smut balls, or spore masses, soil clods, plant debris, etc. (Mathur and Kongsdal, 2003). Visual and stereo-binocular examination of seeds results in detection of exotic pathogens in imported crop germplasm. Some fungal disease symptoms such as ergot of wheat (Claviceps purpurea), Karnal bunt (Tilletia indica) and hill bunt (T. carries/ T. foetida) of wheat; kernel smut (T. barclayana) of rice; downy mildew (Peronospora manshurica) of soybean, etc. (Figure 2).
Figure 1: Quarantine testing procedure for pathogen-free release of exotic crop germplasm
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Figure 2: Symptoms of seed-borne fungal diseases in different crops; a) Claviceps purpurea; b &
c) Karnal bunt and hill bunt of wheat; d) kernel smut of rice; e) downy mildew of soybean Washing test: The washing test is commonly employed to detect the presence of fungal spores adhering to seed surface. The method is quick and can be employed for detecting various types of fungal spores such as conidia, oospores (downy mildews), teleutospores/
uredospores (rusts), smut/bunt spores, etc. (Mathur and Kongsdal, 2003). For detection of Puccinia helianthi/ P. carthami in sunflower/ safflower, the seeds are taken in a test tube containing 10 ml water and stirred on shaker. The suspension is transferred in Petri dish and observed after sedimentation for the presence of spores under stereo-binocular microscope (Figure 3).
(a) (b)
Figure 3: Detection of teliospores of sunflower rust (a) and safflower rust (b)
Seed soaking method: For detection of bunt (Tilletia barclayana) in rice, seeds are soaked overnight in 0.2 per cent sodium hydroxide and examined. The infected seed shows shiny jet black discolouration (Mathur and Kongsdal, 2003). Infected seeds rupturing in a drop of water, releases a stream of bunt spores (Figure 4).
Figure 4: NaOH soaking procedure for detection of paddy bunt
Page | 156 Incubation Method: Incubation is a simple method commonly used for detection of mycoflora accompanied as mycelium, spores, or fruiting structures capable of growing on the seed during incubation of seed on wet blotter or agar (Mathur and Kongsdal, 2003). Surface sterilization of the seeds, using a 4% NaOCl solution, is carried out before incubation to eliminate fast growing saprophytes, if the seeds are heavily contaminated.
1. Blotter test: Blotter test, generally referred as the standard blotter test, is the most efficient means of detecting a large number of seed-borne fungal pathogens. Visually discoloured, deformed and unhealthy looking/ suspected seed are undergone for blotter test by placing the seeds on 3 layers of moist blotter paper in plastic petriplates with proper labelling and incubated at 22±2oC under light in alternate cycles of 12 h light and darkness for 7 days and examined on 8th day under stereo-binocular microscope for presence of seed-borne fungi (Mathur and Kongsdal, 2003). The critical microscopic examination enables the observation of pathogens as developed on their hosts in situ, undisturbed and in a condition of natural growth. The identification is confirmed up to species level by making slides for examining the structure, size and colour of fruiting bodies/ conidiophores/ conidia under compound microscope at different levels of magnification i.e. 4.0 X to 40.0 X (Akhtar et al., 20014). A critical stereo-cum-compound microscopic examinations of seeds on 8th day after incubation results in detection of many seed-borne pathogens (Figure 5). Major detection includes Fusarium nivale on wheat, Phoma lingam, Diplodia maydis and D. macrospora, Phomopsis longicolla, Xanthomonas campestris pv. campestris, Phomopsis helianthi, Dendryphion penicillatum, etc. on different crop germplasm (Akhtar et al., 20016, 2017; Singh et al., 2018).
2. Agar plate method: Like blotter method, agar plate method is also a standard procedure for detection of many seed-borne fungi/ bacteria. Identification of fungi is based on growth and colony characteristics on nutrient mediums like water agar, malt extract agar, potato dextrose agar. Seeds are surface sterilized before plating on the medium and plates are incubated under conditions similar to blotter method. Identification of fungi is based on the macroscopic examination of colony growth pattern and colour. The identification is confirmed by preparing slides and examining under compound microscope for characteristics of mycelium, spore and conidia.
For detection of bacteria, the surface sterilized seeds are placed on nutrient agar medium in Petri plated and incubated at 22–25°C. The bacterial growth will appear on the seed, if infected. Then the associated bacterium is isolated, purified and identified based on morphological, cultural and biochemical tests, etc. Several selective / semi-selective media have been developed and used for detection of specific bacteria associated with seeds.
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e f g h
Figure 5: Detection of seed-borne fungal and bacterial pathogens in different crops; a) Fusarium nivale on wheat; b) Phoma lingam on cabbage; c) Diplodia maydis on maize; d) Diplodia macrospora on maize; e) Phomopsis longicolla on sunflower; f) Xanthomonas campestris pv. campestris on cabbage; g) Phomopsis helianthi on safflower; h) Dendryphion penicillatum on opium.
Molecular detection of pathogenic fungi/ bacteria
1. Loop-mediated isothermal amplification based detection of Colletotrichum capsici: A LAMP assay targeting the β-tubulin sequence for visual detection of C. capsici was developed, (Kandan et at., 2016) The LAMP reaction was optimal at 65°C for 45 min. The results showed that the colour of LAMP products changed from orange to fluorescent green when C. capsici was detected with SYBR Green I (Figure 6). All amplified products from C.
capsici showed bands, while no amplicons were detected for other fungal species and negative control.
Figure 6: Specificity of LAMP assay for C. capsici detection. (left) colour changes of LAMP amplification product after adding 5 ìl 10X SYBR Green I and (right) Agarose gel (1.5%) electrophoresis of 5 ìl LAMP products. M indicates 3 Kb DNA ladder (fermentas), 1 & 2 – C. capsici, 3-Alternaria alternata, 4-Bipolaris rostrata, 5-Fusarium verticillioides, 6-Fusarium solani, 7- Macrophomina phaseolina, 8-Aspergillus niger and 9-Negative control.
Page | 158 2. PCR-based detection of Xanthomonas campestris pv. campestris: A set of rpf region- based primers namely rpfH_F and rpfH_R were developed for Xanthomonas campestris pv.
campestris. The annealing temperature for primers were optimized at 60oC and specific bands of 304 bp for X. campestris pv. campestris were obtained in PCR (Figure 7). The detection sensitivity of the primer pairs was performed by dilution of genomic DNA and results revealed that it could detect up to 0.1 ng μl-1 of template DNA of the pathogen (Kiran et al., 2019; Kumar et al., 2019). This primer is specific to X. c. pv. campestris and there is no cross amplification with other related fungal and bacterial pathogens.
Figure 7: PCR amplification for X. campestris pv. campestris isolates using specific primers rpfH_F and rpfH_R. Lane M- 1 Kb plus DNA ladder; Lanes 1 to 11- X. campestris pv. campestris isolates, Lane C- Negative control.
In addition to conventional diagnostic tools, LAMP assay and PCR-based molecular are substantial in detecting and intercepting large number of pathogens of quarantine significance on a wide range of crops from different sources/ countries which emphasizes the pivotal role of plant quarantine in minimizing risk of introduction of exotic pathogens associated with PGR importing into the country, otherwise huge losses would have occurred due to exotic pathogens/ diseases.