1. Introduction
Sugarcane (
Saccharum officinarum L.) is widely cultivated in the tropics and sub-tropics. It is one of the main sources of sugar in the world [
1]. Sugarcane covers approximately 26.3 million hectares of the global arable land. The total production of approximately 1.9 billion tons [
2]. In Bangladesh (North West and South East of Bangladesh), sugarcane is cultivated on 0.11 million hectares of land for white sugar, ethanol, juice production, chewing, and brown sugar [
3]. Sugarcane plants are susceptible to several diseases, of which red rot which is caused by
Colletotrichum falcatum is one of the devastating diseases (Glomerallacae of Ascomycota).
Glomeralla tucumanesis which is the sexual stage of
C. falcatum [
4], is also called as
Physalospora tucumanesis [
5]. Sugarcane red rot is endemic in the tropics and subtropics and it poses a serious challenge to sugarcane production in Bangladesh [
6]. Depending on cultivars, environment, and pathogen strain, it reduces sugarcane weight up to 29% with sugar recovery loss up to 31% [
7].
Although several studies had been carried out on the biochemical, physiological, molecular analysis of pathogen-plant interactions and the complete genetic basis for the progression of the disease is yet to be identified. To establish novel techniques for the successful control of the red rot disease, a clearer understanding of the phylogenetic relationship and genetic diversity are essential. Currently, the viable way of managing the disease is using resistant cultivars [
8]. However, the rapidly evolving
C. falcatum fungus contributes to the formation of new virulent strains, complicating the red rot resistance development [
9]. The red rot epiphytotic had negatively impacted the widely known cultivars [
10]. Frequent shifts in the pathogen’s genetic structure and incremental shortening of the genetic link of resistant origins are the main factors of the natural choice for new, potential isolates and deteriorate the resistance in the host [
9].Appropriate detection and classification of isolates in
C. falcatum tainting are crucial for the exact taxonomic identification required for disease management and breeding for resistance [
11].
Detection and characterization of
C. falcatum are carried out based on morphological characteristics such as conidia shape and size, setae or teleomorphic appearance, and characteristics of culture such as colour, rate of growth and texture [
12]. However, in different environment
Colletotrichum species can demonstrate morphological and physiological differences. Therefore, accurate identification of
Colletotrichum species is difficult [
13]. On the other hand, molecular marker techniques have improved identification precision, speed, and classification of phytopathogenic fungi [
14]. Nucleotide sequences differ from species to species because of this, rDNA-Internal transcribed spacer (rDNA-ITS) region is commonly used for
Colletotrichum spp. differentiation for various plants [
15]. Phylogenetic analysis of the DNA sequence data from the gene regions of ITS-rDNA, Beta-tubulin (
β-tubulin), Actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been used to construct species-specific primers for
C. acutatum and
C. gloeosporioides species complex detection and phylogenetic analysis [
16]. Knowledge on the variability of pathotypes is significant in selecting the correct isolates for resistance testing in plant breeding programmes. Over the past few decades, molecular markers have been used to determine genetic variation, genetic architecture, and virulence in plant pathogen populations. Because the genetic makeup of
C. falcatum isolates is not known, knowledge on the genetic variability of the population is important for understanding the evolutionary process and prospects to evolve for environmental change [
17]. In Asia, the characterizations of
C. falcatum for sugarcane was carried out in India using sequence analysis of ITS region and genetic diversity by inter-simple sequence repeat (ISSR), Kumar et al. [
9]. Currently, there is dearth of information on red rot disease for sugarcane plantations in Bangladesh. This study aimed to isolate
C. falcatum from infected sugarcane plantations in Bangladesh. Also, the phylogenetic characteristics and the genetic diversity among the populations of
C. falcatum isolates were determined. The results from this study could be a platform for generating detailed information on the genetic variation of sugarcane red rot disease in Bangladesh using molecular approaches. Besides, this research provides valuable information on how to overcome the disease. Thus, the overall findings of this present study could contribute to improving sugarcane production in Bangladesh.
4. Discussion
Colletotrichum falcatum causes red rot disease in sugarcane. This disease is transmitted from one location to another by infected sugarcane [
51]. Several measures have been developed to effectively control the disease, but the measures have not successful. One of the major reasons is due to fact the pathogen develops new races to easily infect existing sugarcane cultivars [
9]. Thus, many popular sugarcane varieties such asIsd 17, Isd 18, Isd 28 and Isd 32, have been withdrawn from commercial fields in Bangladesh [
52]. This is the first study to determine the genetic variation, population structure of
C. falcatum isolates and their race prevalence in Bangladesh. Forty-one
C. falcatum isolates were collected from different districts of the four regions in Bangladesh after which their morphological, virulence, phylogenic relationship and genetic diversity were determined.
Morphological and colony differences indicated that there were significant differences among the isolates. The isolates with whitish-greyand greyish-white colony colour were mostly fluffy; few of them possess flat topography and they produced medium to high sporulation. The grey and dark grey isolates were mostly flat, although some demonstrated raised fluffy and produced low to medium sporulation. The findings are comparable to those of Kaur et al. [
23], who also reported that the isolates had a whitish grey and greyish white color colony which is mostly fluffy and produces medium to high sporulation and the colonies were grey, dark grey, and less fluffy. In a related study, Prema et al. [
22] reported significant difference in the morphology and cultural characteristics of
C. falcatum. The 41 isolates demonstrated difference in mycelial growth rate and these findings are consistent with that of Viswanathan et al. [
53] who focused on nine main pathotypes of
C. falcatum. In terms of conidial morphology, the 41 isolates produced falcate shape; hyaline conidia and the conidial size were similar to the results of Mishra and Behera [
54], Sangdit et al. [
21] and Prema et al. [
22]. Although,
C. falcatum isolates are grouped based on colony and morphological characters, these approaches are not reliable or consistent because of the fungal colony and morphological characters are affected by environmental factors. Moreover, morphological data of
C. falcatum such as growth rate and sporulation correlate weakly with the frequency of the disease infection on sugarcane [
55,
56]. The Pathological assessment revealed that the
C. falcatum isolates induced red rot disease symptoms on sugarcane variety, Isd 28 through artificial inoculation with different severity. Findings revealed that, they were clustered into three virulence cluster namely LV(less virulent), MV (moderately virulent) and V (virulent). The isolates from MV and LV clusters are widely spread in the four major sugarcane growing regions in Bangladesh. However, the isolates from Vcluster are mostly distributed in the sugar mill zone.
Colletotrichum falcatum belongs to an anamorphic fungus and because of this, the virulence variability within populations might have occurred through mutation, heterokaryosis, hybridization and adaption [
55]. Other possible reasons could be variation in the production of hydrolytic enzymes (pectinolytic and cellulolytic) and melanin during host-pathogen interaction. In Bangladesh, susceptible sugarcane varieties/cultivars are widely planted by smallholder. This enables the pathogen to evolve and diversify its virulence because of the high proliferation rate and dispersion capacity. Cluster analysis revealed that there was a weak correlation between virulence level and genetic diversity amongst
C. falcatum isolates (
Table 4 and
Figure 8). This finding corroborates the previous reports pertaining the virulence level of pathogens isolated from sugarcane and geographical distribution [
23,
28,
57].
The final sequence of ITS,
β–tubulin, actin, and GAPDH genes were identified as
C. falcatum and revealed high similarity to the reference sequences in the Genbank database. These results are consistent with the findings reported by Sangdit et al. [
21] for molecular identification of
C. falcatum, Mahmodi et al. [
58] and Aktaruzzaman et al. [
59] for
C. tuncatum. Sequences of the ITS,
β-tubulin, actin, and GAPDH have been widely used to determine the phylogenetic relationships among many
Colletotrichum species which had clarified the taxonomic relationships within the genus [
51,
60]. In this present study, ITS,
β-tubulin, actin, and GAPDH genes sequences data were used in combination with the first time for phylogenetic analysis of
C. falcatum that contributed to the taxonomic study in Bangladesh and the world as a whole. Furthermore, the accuracy of molecular identification is high and more reliable compared with the conventional methods to especially classify different genus of
Colletotrichum which are closely related species [
14,
61,
62]. The traditional method is less successful due to the assumed presence of intermediate forms between species, morphologic plasticity, and overlapping phenotypic characters. In contrast the molecular biology technique encompasses alternative and supplementary methods for overcoming the difficulties in identifying up to species level [
63]. The molecular method is reputed for enabling rapid identification of isolates and it also clarifies the relationships between fungal organisms [
64].
The genetic relatedness and ancestry of
C. falcatum were determined and the four examined genes used were successfully differentiated. The collection of molecular entries of
C. falcatum in the NCBI database were relatively limited. Thus, selected
C. falcatum isolates were compared to Bangladesh isolates to determine the genetic relationship of
C. falcatum isolates worldwide. The comparison was carried out based on the ITS,
β-tubulin, actin, and GAPDH sequence data to identify the phylogenetic relationship of
C. falcatum isolates. Evolutionary history through maximum likelihoodtree based on four genes inferred that there was no geographical structure in the distribution of
C. falcatum isolates among the different sites in Bangladesh. Four gene sequences also revealed that
C. falcatum isolates from Bangladesh differed from reference
C. falcatum isolates from other countries (India, China, Thailand, Mexico, USA, Japan, and the Netherlands) because of the variation of nucleotide in different loci positions. The Actin and GAPDH genes phylogenetic analysis showed that several
C. falcatum isolates from India, one from China, and one from Thailand, were cluster together with the Bangladesh isolatesThis is related to the fact that: (i) India, China, and Thailand are geographically close to Bangladesh and (ii) Planting materials are exchanged among Southeast Asian countries including India, China, and Thailand. This assumption is supported by the findings of Oghenekaro et al. [
65]. Four genes phylogenetic tree comparison results demonstrated that
C. endophytum was the closest species to
C. falcatum and this observation is consistent with that of Hyde et al. [
66], who stated that
C. endophytum is a sister taxon of
C. falcatum.
ISSR-PCR fingerprinting is a valuable method for population structure studies and differentiating individual fungal isolates [
67]. ISSR-PCR is more accurate than the RAPD-PCR variant for determining the genetic variation of the individual isolates [
68,
69]. In this present study, 10 selected ISSR markers were used in the genetic diversity analysis against 41
C. falcatum isolates. These ISSR markers gave different reproducible bands, and the high percentage polymorphism suggests genetic variation among the isolates. The different band pattern generated by ISSR markers in the study could be attributed to (i) intraspecific variation: ISSR fingerprinting is higher for certain fungal species and lower for others because of intraspecific variations among the markers [
60],(ii) abundant in (CA)n and (GT)n content: this may clarify the more significant number of alleles reported within those loci and the more polymorphic ISSR-PCR bands [
69], (iii) primer-binding site: absence of a primer-binding site due to mutationin priming site, strand slippage during DNA polymerization due to template instability, deletions or insertions that might decrease orincrease the amplifiedfragment length sufficiently to be scored as a separate locus, and structural rearrangements of the chromosomes that prevent polymerization [
70], and (iv) base composition: The base content of ISSR primers influence the quality of fingerprints, as primers having higher GC content (>50 percent) consequence in background noise and non-homologous co-migrating fragments, while pure A or T primers resulted in amplification of the products. Adjusting the annealing temperature of primer and other conditions of reaction reduces these effects [
71].
The genetic diversity of
C. falcatum was higher at the species level than at the population level in this study. Among the four populations of
C. falcatum, different (highest to lowest) genetic diversity was evident. Variations of genetic diversity among the populations might have resulted from some factors. These factors are as follows: (i) population size: larger populations maintain greater genetic diversity under neutrality; (ii) age of population: Aged populations have a more extensive genetic diversity than the newly colonized habitat, mainly if only a few colonizers form this population. The older population could have mutational events to add new genetic variants and for genetic drift to increase the prevalence of these alleles to measurable levels; (iii) location: the isolates located at or near the center of origin from which the species originate have a higher degree of gene diversity than the isolates from other areas because the original population are older [
72,
73,
74]. Weeds et al. [
74] illustrated that the genetic variance in
C. gloeosporioides isolates are higher where native or naturalized host species occur compared with areas where the host species have recently been introduced.
The evidence of admixture of some of the
C. falcatum isolates from the four populations occurred in this present study. This occurred because of climatic conditions of these locations were similar. The similar climate enabled the isolates to co-exist. Other factors such as spontaneous natural mutations, genetic drift, gene flow, crop rotation, sampling year, and alternate host might have played significant role in terms of variation among the isolates of
C. falcatum [
70,
75,
76,
77,
78]. Furthermore, molecular markers are more informative than rDNA-ITS sequencing in terms of detecting the diversity within the individual population but less successful in detecting the variation between different populations [
79].