Molecular Variability of Colletotrichum spp. Associated with Anthracnose of Soybean

Soybean (Glycine max L.) is one such protein crop that has high economic importance across the globe due to its usage as food, feed and as by-products in industrial sector. It is very well known for its high protein and oil content accounting to nearly 40 and 20 per cent respectively and hence known as “Golden Nugget”. This crop is a source of vegetable oil, proteins for human and animal feeds and is grown throughout the tropical, sub-tropical and temperate regions. The crop coverage is about 125 million hectare in the world with a production and productivity of 358.85 million tons and 2870 kg/ha respectively. Major soybean producing countries include USA, Brazil, Argentina, China and India. In India, it is cultivated over an area of 11.30 m ha, with production of 10.93 mt and productivity of 960 kg/ha, wherein the Madhya Pradesh state is a massive producer and thus obtaining the title as “Soya State”. Followed by Madhya Pradesh is the Maharashtra, Rajasthan, Karnataka and Telangana. Due to its multifaceted uses it has been an increase in production, but still face biotic and abiotic constraints during the production practices. Anthracnose in particular, imposes a major threat as it infects the plants and seeds, which would lead to a yield loss upto 100 per cent (Yang and Hartman, 2016).
       
Nakata and Takimoto (1934) disclosed for the first time about anthracnose leaf spot of soybean from Korea during 1917. Colletotrichum species can infect soybean at all stages (Sharma et al., 2011) and is associated with wide number of species, of which C. truncatum is the most commonly associated species (Sharma et al., 2011; Dias et al., 2018). Symptoms manifested after infection on the crop includes chlorotic dull brown lesions, which coalesce and turn to dark brown and necrotic, thus leading to premature drying of foliage. Cankerous lesions can also be observed on the infected petiole and stem region. Lesions are also recorded on infected pods where they appear concentric, wherein severely affected pods bear chaffy and shrivelled seeds (Yang et al., 2015; Uddhav, 2017). C. truncatum is the widely associated species in all soybean growing countries. In India, soybean anthracnose associated with C. truncatum was first reported by Verma and Upadhyay (1973). The other species associated with soybean anthracnose includes, C. gloeosporioides, C. incanum and C. dematium, C. chlorophyti, C. brevisporum, C. musicola, C. plurivorum and C. sojae. So, this study aims to identify the association of Colletotrichum species with soybean anthracnose in India. As, there is limited information on variability of C. trunactum and diversity of species infecting soybean.

During 2019, twenty-eight fields were sampled for anthracnose-infected soybean, which includes, Karnataka, Maharashtra, Madhya Pradesh, Uttarakhand, Nagaland, Jharkhand and Rajasthan. The samples were washed; dried and the pathogen was isolated using the standard tissue isolation method (Rangaswamy and Mahadevan, 1972) on sterilized Potato Carrot Agar (PCA) medium, subsequently incubated at 28^oC. Sub-culturing was carried out to purify the cultures and was maintained on Potato Dextrose Agar (PDA) slants for further use. Phenotypic characterization was carried out with respect to twenty-eight isolates collected from different locations (Table 1) were cultured on Potato Carrot Agar (PCA) medium for sporulation. The mycelium and spores of all the isolates was examined under the microscope for morphological characters.
 

       
To prove the pathogenicity, spore suspension (1 × 10⁶ spores per ml) of all isolates was prepared using sterile distilled water. Forty days old seedlings of soybean, which is susceptible to anthracnose, i.e., JS35, was used, upon which the spore suspension was sprayed. The seedlings were covered with polythene bags for 48 hours to provide favorable condition for infection to commence. The plants were regularly observed for symptom development.
       
The genomic DNA of all the 28 isolates was carried as per Murray and Thompson (1980) with slight modification. Mycelium from 4 to 5 days old pure broth culture was harvested using sterile Whattman filter paper and transferred to pre-chilled pestle and mortar. The mycelium was homogenized to fine powder using liquid nitrogen and about 100 mg of the fine powder was transferred to pre-sterilized micro-centrifuge tubes containing freshly pre heated CTAB buffer. 10 microlitre of beta-mercaptoethanol was added to each tube and mixed well by inversion. The tubes were incubated in hot water bath at 65^oC for 45 minutes with intermittent mixing of the tubes. After incubation, equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) was added and mixed well. The tubes were centrifuged at 10000 RPM for 10 minutes. The upper aqueous phase was collected into fresh tubes to which equal volume of chloroform: iso-amyl alcohol was added and mixed. Later, the tubes were centrifuged at 10000 RPM for 10 minutes in order to obtain the three phases in the tube, from which, the upper aqueous phase was collected and transferred into fresh micro-centrifuge tubes. Equal volume of pre-chilled iso-propanol followed by mixing and incubation at -20^oC overnight. The tubes were centrifuged at 10000 RPM for 20 minutes and decanted to retain the pellets. The pellets were washed with 70% ethanol, air-dried and dissolved in TE buffer and stored at -20^oC for further use.
       
Molecular detection was carried out using primers complementary to 5.8S RNA gene with the flanking ITS, namely ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') (White et al., 1990). The PCR cocktail was prepared, which consists of 2 mM MgCl₂, 200 µM of each dNTP’s and 2.5 U of Taq polymerase (Takara, Japan) in a 0.2 ml micro-centrifuge tube and to each tube, 50 ng of DNA was dispensed to make a final volume of 20 µl. The tubes were vortexed very gently. PCR was performed in a thermocycler with conditions as: Initial denaturation of 94^oC for 5 minutes, 32 cycles each of denaturation at 94^oC for 50 seconds, 52^oC for 45 seconds and extension of 72^oC for 1 minute, followed by one cycle of final extension of 72^oC for 5 minutes. A water control (no template control) was maintained along with the twenty-eight samples.
       
Nucleotide sequences of the isolates were subjected to BLAST analysis for homology search in the NCBI platform and all the twenty-eight sequences were deposited in GenBank of NCBI and accession numbers assigned were obtained (Table 1). Phylogenetic analysis for the 28 isolates was executed using MEGAX tool. Further, a maximum likelihood tree was developed to assess the relationship within these isolates.

Symptoms noticed in anthracnose infected soybean plants under field situation was, light to brown colored lesions of which, some appeared necrotic which gave rise to blighted condition in severely affected plants. During the microscopic study made, the conidia of SeMP22 were aseptate and cylindrical in shape, however truncate conidia were observed for the other 27 isolates. In common among all the isolates was the cup shaped acervuli (Plate 1). On PCA medium, vegetative mycelium initially appeared white which subsequently turned to greyish colour (Plate 2). The mycelium appeared hyaline, septate and branched. During pathogenicity test, symptoms produced were noticed initially at 6 days after inoculation, which were similar to those found in field conditions (Plate 3). The infected pods were malformed and chaffy. The pathogen was re-isolated and observed for morphological characters, which were similar to Plate 1. Several evidences revealed the symptoms of soybean anthracnose as appearance of necrotic lesions on leaf that leads to premature drying, formation of concentric rings on pods with acervuli as sign of infection and also the emergence of necrotic spots on petiole and stem (Nagaraj et al., 2014; Yang et al., 2015; Uddhav, 2017, Yang and Hartman, 2016). These instances give the evidence that formation of such symptoms being associated with Colletotrichum spp.
 

 

 

       
The microscopic observations made for spores were similar to that described earlier (Plate 1). Such similar results of morphological characterization under microscope were discussed by various researchers (Roy, 1996; Photita et al., 2005). Association of C. plurivorum was noticed whose morphology was also described by de Silva et al., (2019) as cylindrical conidia without septa and hence this was confirmatory with our present findings.
       
In the molecular detection, all the isolates amplified at 550 bp, which was measured using the 1Kb DNA marker (Plate 4). On contrary there was no amplification foreseen in the no template control (NTC). Among the 28, 18 isolates were obtained from different locations of north Karnataka, where soybean was extensively grown. During the homology search, they showed similarity of more than 98 per cent to C. truncatum in the NCBI database, of which some reported from different host other than soybean (Table 1). Isolates namely JMP19, MMP20, SMP21 and SeMP22 (Table 1) were collected from parts of Madhya Pradesh showed similarity to Colletotrichum species. However, the isolate, SeMP22 was 99.82 per cent similar to C. plurivorum reported on papaya, while the other isolates showed more than 99 per cent homologous with C. truncatum. Isolates collected from Maharashtra, namely, LMH23 and AMH24 showed 100 per cent homology to C. truncatum reported on Capsicum annuum (Table 1). The other isolates namely, KRJ25 from Rajasthan, RJH26 from Jharkhand, NNL27 from Nagaland and PUK28 from Uttarakhand were more than 99 per cent homology to C. truncatum.
 

       
A maximum likelihood tree was constructed (Fig 1) for the twenty-eight sequences using the MEGAX bioinformatics tool. The analysis showed formation of two main clusters, in which Colletotrichum plurivorum (SeMP22) obtained from Madhya Pradesh (Sehore) solely belonged to one cluster and all the other 27 C. truncatum isolates were grouped together to form another cluster.
 

       
To date, this is the first report of C. plurivorum infecting soybean in India. C. plurivorum was initially known as C. sichuanensis, which infected Capsicum annuum in Sichuan Province of China, later, it was regarded as C. plurivorum as a distinct species with a wide host range (Damm et al., 2019). Recently, it has been reported to cause anthracnose in papaya (Sun et al., 2019), okra (Batista et al., 2020) and in chilli (Saktivel et al., 2018).

Soybean [Glycine max (L.) Merill] is an internationally recognized oil seed crop, which gets infected by several fungal diseases, out of which Colletotrichum truncatum causing anthracnose or pod blight is an economically important disease. In our study, we hereby report the infection of soybean with Colletotrichum plurivorum, which has not been reported earlier in India. Soybean anthracnose is associated with wide number of pathogenic and endophytic Colletorichum sps. In the present investigation we report the association of C. truncatum and C. plurivorum in soybean.

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