**4. Diagnosis**

Identification of *Colletotrichum* species based on morphological characteristics (size and shape of conidia; presence of setae) and colony characteristics is generally used by several workers [56–59]; it is widely used in seed health testing labs for detection of *C. capsici* in germplasm for pest free conservation of chilli seeds [21]. As the pathogen is seed-borne, there is threat of introduction of this pathogen along with import of germplasm (including Chilli) from different countries; therefore, while importing from any other country, there is a need to examine the samples very critically including sensitive molecular diagnostic tools to prevent entry of this pathogen associated with germplasm [60]. Moreover, for the accurate identification of the pathogen at species level molecular methods are widely adapted. Loop-mediated isothermal amplification (LAMP) assay was used for the accurate and sensitive detection of *C. capsici* LAMP primers (β-tubulin gene sequences based) were designed and it was reported that it could detect as little as 10 fg/μl of *C. capsici* pathogen in comparison with only upto 1 ng/μl of *C. capsici* detection using polymerase chain reaction [61]. A sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of *C. capsici* in chilli seeds and fruits. This markers did the amplification of an expected 250-bp fragment from genomic DNA and these markers were very much sensitive as it was reported that the marker could detect purified *C. capsici* DNA template up to 1 pg and DNA from *C. apsici* infected

chilli fruits up to 25 ng [59]. As these two markers are very sensitive, these may be very useful in detection of the pathogen in imported germplasm in plant quarantine laboratories.

COL1/COL2 primers were used for amplification of the specific internal transcribed spacer region of tested *Colletotrichum* species (*C. acutatum*, *C. truncatum* and *C. gloeosporioides*) with a specific band of 460 base pairs. *C. gloeosporioides* was detected at a low level of 1000 conidia on chilli leaf and fruit by this primer [62]. Another, primer set based on the sequences of the ribosomal internal transcribed spacer (ITS1and ITS2) regions of *C*. *truncatum* was designed and standardized for the detection of *C*. *truncatum* in infected plant tissues using PCR assay. The sensitivity was 10 pg of genomic DNA from the pathogen [63]. Machenahalli et al. [64] detected pathogens from different parts of plant like seeds, fruits, infected twig/stem by PCR-based method by using specific primers. *C. truncatum* was amplified by species specific primer (C.cap-f and C.cap-r) as single band at 450 bp. *C. gloeosporioides* was amplified by species specific primers CgInt at 450 and *C. acutatum* by CaInt at 490 bp respectively. The accurate identification is very important for choosing the correct management strategy for this disease.
