Morphological and molecular characterisation of Colletotrichum gloeosporioides (Penz) isolates obtained from Dioscorea rotundata (Poir)

Anthracnose disease is a major constraint to yam production in tropical West Africa and anywhere the crop is cultivated. This study determined the cultural characteristics and growth rates of mycelia and also characterised 6 isolates of Colletotrichum gloeosporioides, the causal agent of the yam anthracnose disease, obtained from Dioscorea rotundata leaves, vines and setts in the Tolon District of Ghana. The cultural characteristics and mycelial growth rates of the isolates were determined on Potato Dextrose Agar (PDA). The C. gloeosporioides isolates were characterised using polymerase chain reaction technique with the universal primer pairs ITS1/ITS4 and NS1/NS2, C. gloeosporioides species specific primer pairs CgInt/ITS4 and CgLac-f/CgLac-r, and C. acutatum species specific primer pairs CaGlu-f1/CaGlu-r1 and Ca-f1/Ca-r1. Based on the PCR, six isolates of C. gloeosporioides with distinct cultural characteristics were obtained. There were no significant differences (P ≤ 0.05) in mycelial growth rates among the isolates. The C. gloeosporioides isolates produced characteristic band sizes on ITS1/ITS4, NS1/NS2, CgInt/ITS4 and CgLac-f/CgLac-r. None of the isolates produced a band on CaGluf1/CaGlu-r1 and Ca-f1/Ca-r1. The proper identification of C. gloeosporioides, the pathogen responsible for the D. rotundata anthracnose is important for the proper management of the disease.


INTRODUCTION
Yam, a staple crop is mainly cultivated in the tropical and subtropical regions for its tubers (Agrios, 2005;Achar et al., 2013). Unfortunately, these regions also provide favourable conditions that support the growth and survival of the yam anthracnose disease pathogen, Colletotrichum gloeosporioides (Penz); a major threat to yam production worldwide (Agrios, 2005;Chaube and Pundhir, 2009;Lebot, 2009;Reddy, 2015). In West African yam growing countries, the most important commercial yam species such as D. alata and D. rotundata are susceptible to the anthracnose disease (Ayodele et al., 2000;Lebot, 2009 (Demuyakor et al., 2013), with the former being the most preferred (Otoo et al., 2015). During cultivation, the disease affects the yam leaves and vines, and severe infection results in yam plant defoliation and vine dieback (Ayodele et al., 2000). C. gloeosporioides has also been reported to cause an orange-brown yam tuber rot known as "dead skin" (Green and Simons, 1994;Reddy, 2015).
Yams are mainly cultivated from yam setts, which if not obtained from certified sources could be infected with C. gloeosporioides which is capable of initiating anthracnose disease on yam crops (Aighewi et al., 2003;Asiedu and Sartie, 2010;Ayoola, 2012;Osei-Adu et al., 2016). C. gloeosporioides is not host specific and as such the presence of other susceptible crops in and around yam fields could also be a source of inoculum for the establishment of the disease on yam crops (Lebot, 2009). The spores of C. gloeosporoides require moisture, optimum temperature (20 -30°C) and high relative humidity to germinate and establish on yam crops, with mature spores mainly disseminated by rain splash (Sharma and Kulshrestha, 2015).
To enhance yam production, there is the need to manage the anthracnose disease on the crop. The proper identification of the causative agent of the disease is crucial for appropriate disease management. Identification of C. gloeosporioides based on cultural, mycelial growth rate and morphological characteristics can be confused with other species within the genus, more especially C. acutatum (Chowdappa and Kumar, 2012;Reddy, 2015). According to Serra et al. (2011), different species of Colletotrichum are capable of infecting a single host. The foliage infection of C. acutatum and C. gloeosporioides are difficult to differentiate in terms of their symptoms and cultural morphology (Shi et al., 2008). This makes it difficult to differentiate between C. acutatum and C. gloeosporioides; hence the need to use molecular techniques for the proper identification of Colletotrichum isolates (Serra et al., 2011). The polymerase chain reaction (PCR) technique has been documented as one means of properly identifying C. gloeosporioides isolates (Shi et al., 2008;Serra et al., 2011;Raj et al., 2013;Chagas et al., 2017). The proper identification of the D. rotundata anthracnose disease pathogen is essential for selecting appropriate strategies in managing the disease to enhance the crop"s productivity. This study sought to determine the cultural characteristics, mycelial growth rates and molecular characteristics of C. gloeosporioides isolates obtained from infected D. rotundata leaves, vines and setts in the Tolon district of Ghana.

Study site
The study was conducted in the Spanish Laboratory at the Nyankpala Campus of the University for Development Studies, during the 2016 and 2017 main cropping seasons. The site which is located in the Tolon district of Ghana (latitudes 9° 15ʹ and 10° 02ʹ North and Longitudes 0° 53ʹ and 1° 25ʹ West), were the D. rotundata leaves, vines and setts with anthracnose symptoms were obtained for the study. The mean annual rainfall ranges from 950 to 1,200 mm and humidity between April and October can be as high as 95% in the night, falling to 70% in the day. The soil is generally of the sandy loam type and the vegetative cover is basically Guinea Savanna interspersed with short drought resistant trees and grassland.

Sampling of anthracnose infected D. rotundata plant parts
Ten anthracnose infected D. rotundata leaves were randomly obtained from each of 48 yam farms in the Tolon district. Infected D. rotundata vines (a total of 5) were obtained by a complete survey of each of the 48 farms for die-back symptoms. One hundred and fifty D. rotundata planting setts (5 per farmer) were randomly obtained from 30 yam farmers. These setts were carefully examined and those with "dead skin" symptoms selected for the study. A total of 21 symptomatic "dead skin" setts were obtained for the study. The samples were then conveyed in well labelled envelops to the laboratory.

Preparation of culture media
The medium was prepared as directed by the manufacturer"s (Sigma-Aldrich Co., Spain) recommendation of 39 g of Potato Dextrose Agar (PDA) per litre of distilled water. Two hundred and fifty (250) mg of amoxicillin was added to suppress bacterial growth. The mixture was heated to completely dissolve the solutes and then autoclaved at 1.03 kg cm -2 pressure at 121°C for 15 min. About 20 ml each of the molten media were poured into Petri dishes (9 cm diameter) and allowed to solidify before use.

Isolation of C. gloeosporioides
Fragments (1 cm) of symptomatic tissues consisting of diseased and healthy parts were obtained from the anthracnose infected D. rotundata leaves, and vines affected by die-back. Similarly, 0.5 cm 3 fragments were obtained from portions of the yam setts with the "dead skin" symptoms. These tissues were each washed with tap water and surface sterilised in 70% alcohol for 3 min. The tissues were then rinsed with three changes of sterilised distilled water. Each tissue was inoculated on the PDA and incubated at ambient temperature (28±2°C) for 7 days. The mycelia that grew were subcultured onto fresh PDA, and further sub-cultured until pure cultures of C. gloeosporioides were obtained.

Identification of C. gloeosporioides
The cultural and microscopic view of the morphological characteristics of 7 days old pure culture of the isolates under a compound microscope (Leica DME, Leica Microsystems, Shanghai, China) were compared to those documented by Barnett and Hunter (2006).

Determination of mycelial growth rate
The method of Than et al. (2008) was employed with some modifications. For each C. gloeosporioides isolate, the mycelial growth on PDA plate was measured daily until the fifth day. The

DNA extraction using the CTAB protocol
Extraction of DNA from the fungal mycelia was done according to the CTAB protocol (Lodhi et al., 1994). The mycelia were grinded to a fine paste in 400 μl of extraction buffer in microfuge tubes using a pestle and then incubated in a re-circulating water bath at 65°C for 15 min, followed by centrifugation at 12000 rpm for 5 min. Four hundred (400) µl of supernatant was transferred into new Eppendorf tubes and 250 μl of Chloroform: Iso Amyl Alcohol (24:1) was added to each tube, mixed with the solution by inversion and centrifuged at 13,000 rpm for 1 min. The upper aqueous phase was transferred into a clean microfuge tube and 50 μl of 7.5 M Ammonium Acetate, followed by the addition of 400 μl of ice-cold ethanol to each tube to precipitate the DNA. This was then mixed by slow inverted movements that caused the DNA to precipitate at the bottom of the tubes. The tube containing the DNA was then centrifuged at 13,000 rpm for 5 min after which the propanol was decanted. This was washed twice with 0.5 ml of 70% ethanol and centrifuged at 15,000 rpm for 5 min. The DNA was then dried and 50 µl of TE buffer was added to dissolve it. It was then stored at -20°C until required.

PCR amplification of C. gloeosporioides strains
The reaction volume was 20 µl containing 2 µL of genomic DNA, 2X Master Mix with standard buffer (New England Biolab, UK) and 1 µL of each primer. PCR amplification was started at an initial denaturation step at 94ºC for 5 min followed by 35 cycles of denaturation at 93ºC for 1 min; for each primer pair, DNA annealing was done at a specific temperature (Table 1) for 1 min with extension at 72ºC for 2 min and a final extension at 72ºC for 5 min. The formation and size of the PCR products were checked by electrophoresis in 1.0% agarose gel and stained with ethidium bromide.

Pathogenicity test
This was determined by a modified detached leaf method described by Shivanna and Mallikarjunaswamy (2009) with some modifications.
Fully expanded apparently healthy D. rotundata leaves were detached from their plants and washed with tap water to remove any dust particles on them. The leaves were then surface sterilized with 70% alcohol for 3 min and rinsed in three changes of sterile distilled water and left to air-dry in a microflow laminar flow workstation. A leaf was then placed on moistened blotter discs (filter paper) in a Petri dish, wounded by gentle pricking with a sterilized needle and inoculated with 1 ml spore suspension (2 x 10 6 spore/ml) of C. gloeosporioides and then incubated under light-dark cycle of 12/12 h at 23±2°C for 7 days. The leaves that served as control were prick inoculated with sterile distilled water. Fungal pathogens were re-isolated on PDA plates and isolates compared with the inoculants culture based on colony and morphological characteristics.

Data analysis
The mycelial growth rate data were subjected to one-way Analysis of Variance (ANOVA) with GenStat (12 th edition) statistical software. Treatment means were separated with the least significant difference of Tukey"s multiple-range test at 5% significance level.

Cultural characteristics and mycelial growth rate of the C. gloeosporioides isolates
Based on the cultural and morphological characteristics, the C. gloeosporioides isolates obtained from the anthracnose infected yam leaves, vines and planting setts were grouped into six (CDr1, CDr2, CDr3, CDr4, CDr5 and CDr6) ( Table 2). Each of the isolates had cylindrical conidia with both ends rounded. Setae were not recorded for the isolates CDr1, CDr2, CDr3, CDr4, and CDr6, except for CDr5. The mycelial growth rate of the C. gloeosporioides isolates ranged from 4.55±0.287 (CDr4) to 5.35±0.263 (CDr3) mm per day (Table 2). There were no significant differences (P ≤ 0.05) in mycelial growth rate among the various isolates (Table 2).

F (pr) 0.291
Means ± standard errors in the same column followed by the same letter are not significantly different (P ≤ 0.05) as determined by Tukey"s multiplerange test. Key: (+) = isolated, (-) = not isolated.

Pathogenicity test
The yam leaves inoculated with the C. gloeosporioides conidia suspension developed symptoms of anthracnose disease. The re-isolation of the pathogen from these diseased leaves on PDA produced similar cultural and morphological characteristics as the mother culture. Anthracnose disease symptoms were not developed on the negative control leaves. These outcomes satisfied Koch"s postulate.

DISCUSSION
The cylindrical shaped conidia with rounded ends recorded for each of the isolates CDr1, CDr2, CDr3, CDr4, CDr5 and CDr6 were similar to the observations made by Abera et al. (2015) who also worked on C. gloeosporioides. The mycelial growth rate per day for the various isolates which ranged from 4.55±0.287 to 5.35±0.263 mm were within the growth rate range of 3.6 to 11.2 mm recorded for C. gloeosporioides (Than et al., 2008;Abera et al., 2015). The insignificant differences (P ≤ 0.05) in growth rate among the C. gloeosporioides isolates confirmed Than et al. (2008) report. Interestingly, Abera et al. (2015) recorded significant differences in mycelial growth rate among C. gloeosporioides isolates. These contradictory reports on significant differences in mycelial growth rate among C. gloeosporioides isolates attested that, it is not reliable for distinguishing among Colletotrichum isolates. The observed variation in colour and pattern of growth among the pure cultures of CDr1, CDr2, CDr3, CDr4, CDr5 and CDr6 agreed with the findings of Than et al. (2008), Gautam (2014), Abera et al. (2015) and Appiah-Kubi et al. (2016) who also documented differences in cultural characteristics among isolates of C. gloeosporioides. The differences in cultural characteristics among the C. gloeosporioides isolates could be attributed to environmental rather than genetic factors. This is because according to Than et al. (2008), the use of morphological and phenotypic characteristics in distinguishing among Colletotrichum species can be deceptive, since different environmental conditions can cause variation among these traits. This makes it unreliable to depend solely on the cultural and morphological characteristics of Colletotrichum species for their identification. According to Cannon et al. (2000), the DNA traits of an organism are not directly influenced by environmental conditions and hence the most reliable method of distinguishing among Colletotrichum species will be the use of molecular techniques such as PCR.
When the DNA extracts of the C. gloeosporioides isolates were subjected to PCR run with the universal primer pairs ITS1/ITS4, the band size amplification of approximately 580 bp was yielded for all isolates. This agreed with the findings of Raj et al. (2013) who also  gloeosporioides isolates were subjected to PCR using the universal ITS1/ITS4 primer pair. Also, the various isolates were not separated when run on NS1/NS2 primer pair, because each of them produced a band size of 560 bp. This agreed with the findings of Shi et al. (2008) who also observed a 560 bp band size for C. gloeosporioides isolates after subjecting them to PCR using the universal primer pair NS1/NS2. The 463 bp detected with the C. gloeosporioides specific primer pair CgInt/ITS4 for isolates CDr1, CDr2, CDr3, CDr4, CDr5 and CDr6 also confirmed all the isolates as the same strain of C. gloeosporioides. A similar observation was made by Shi et al. (2008). Also, Serra et al. (2011) reported that Colletotrichum isolates that produced band sizes on the primer pair CgInt/ITS4 was a confirmation of their identity as C. gloeosporioides. The PCR analysis of the isolates CDr1, CDr2, CDr3, CDr4, CDr5 and CDr6 on the C. gloeosporioides species specific primer pair CgLac-f/CgLac-r produced band sizes for the various isolates; also revealing them as C. gloeosporioides. This agreed with the observation of band size production by C. gloeosporioides on the primer pair CgLac-f/CgLac-r documented by Shi et al. (2008) and Chagas et al. (2017).
The difficulty in distinguishing between C. gloeosporioides and C. acutatum using cultural and morphological characteristics necessitated the PCR run of all isolates on the C. acutatum species specific primer pairs CaGlu-f1/CaGlu-r1 and Ca-f1/ Ca-r1 to further clarify their identity as C. gloeosporioides. The lack of band production by the various C. gloeosporioides isolates when they were subjected to PCR on each of the C. acutatum species specific primer pairs CaGlu-f1/CaGlu-r1 and Ca-f1/Ca-r1 further confirmed their identification as C. gloeosprioides. A similar observation was made by Shi et al. (2008) who also recorded no DNA amplification for the PCR analysis of C. gloeosporioides on each of the C. acutatum species specific primer pairs CaGlu-f1/CaGlu-r1 and Ca-f1/Ca-r1. The pathogenicity test which showed that the C. gloeosporioides isolates produced anthracnose disease symptoms on the yam leaves confirmed the isolates as the causative agents of the disease.
The isolates of this study were identified as C. gloeosprioides based on their cultural and morphological characteristics, as well as mycelial growth rate. The isolates were confirmed as C. gloeosprioides as they produced characteristic bands on the universal primer pairs ITS1/ITS4 and NS1/NS2, and C. gloeosprioides species specific primer pairs CgInt/ITS4 and CgLacf/CgLac-r. Also, the lack of bands amplification by CDr1, CDr2, CDr3, CDr4, CDr5 and CDr6 on the C. acutatum specific primer pairs CaGlu-f1/CaGlu-r1 and Ca-f1/Ca-r1 further confirmed all the isolates as C. gloeosporioides. The proper identification of C. gloeosporioides as the pathogen causing the D. rotundata anthracnose disease would aid in the development of appropriate strategies and tactics in the management of the disease in the Tolon district.