Characterization of Alternaria and Colletotrichum Species Associated with Pomegranate (Punica granatum L.) in Maharashtra State of India

Fungal pathogens are a major constraint affecting the quality of pomegranate production around the world. Among them, Alternaria and Colletotrichum species cause leaf spot, fruit spot or heart rot (black rot), and fruit rot (anthracnose) or calyx end rot, respectively. Accurate identification of disease-causing fungal species is essential for developing suitable management practices. Therefore, characterization of Alternaria and Colletotrichum isolates representing different geographical regions, predominantly Maharashtra—the Indian hub of pomegranate production and export—was carried out. Fungal isolates could not be identified based on morphological characteristics alone, hence were subjected to multi-gene phylogeny for their accurate identification. Based on a maximum likelihood phylogenetic tree, Alternaria isolates were identified as within the A. alternata species complex and as A. burnsii, while Colletotrichum isolates showed genetic closeness to various species within the C. gloeosporioides species complex. Thus, the current study reports for the first time that, in India, the fruit rots of pomegranate are caused by multiple species and not a single species of Alternaria and Colletotrichum alone. Since different species have different epidemiology and sensitivity toward the commercially available and routinely applied fungicides, the precise knowledge of the diverse species infecting pomegranate, as provided by the current study, is the first step towards devising better management strategies.


Introduction
Pomegranate (Punica granatum L.) has been cultivated as a fruit crop since ancient times. It produces edible fruits with innumerable health benefits and high commercial value. Moreover, in recent years, possible applications of extracts of pomegranate fruit peel as natural pesticides or food preservatives have also been envisaged [1][2][3]. Over the last few decades, global market demand of pomegranate fruit has increased remarkably, resulting in alluring monetary returns to growers and a constant increase in cultivation area and production of this horticultural crop, especially in India. Globally, India is the largest pomegranate producer, with more than 41% of the world's area and production. Currently, in India, the crop occupies an area of 288,000 ha, with a production of 3,256,000 tons [4]. Maharashtra state is the first pomegranate producer in India with a cultivated area of 171,000 ha and a production of 1,795,000 MT, as well as the first exporter, with 51,699 MT and a value of INR 3520 million in 2020-2021 [5], with a share of 59.4% in area, 55.13% in production, and 84.41% in export at the national level. In Maharashtra, the largest area cultivated with pomegranate (47,380 ha) is in the Solapur district. Due to the unique properties of pomegranate produced in Solapur, it has been awarded the geographical

Sample Collection and Fungus Isolation
Alternaria and Colletotrichum isolates characterized in this study were isolated from symptomatic fruits and leaves of pomegranate collected from several orchards in different geographical regions of India, such as Maharashtra (MH), Karnataka (KA), Uttar Pradesh (UP), Madhya Pradesh (MP) and Tamil Nadu (TN), during 2015-2021 (Supplementary  Table S1). For sample collection, places that were at least 100-150 km apart were included. Symptomatic tissues were excised, disinfected with 1% sodium hypochlorite (NaOCl) and thereafter rinsed three times with sterile distilled water. The sterile tissues were then placed on sterile potato dextrose agar (PDA, HiMedia Laboratories Pvt Ltd., Mumbai, India) medium with pH 7 and incubated at 24 ± 1 • C. All the isolates were purified by the hyphal tip technique and stored at 4 • C in mineral oil until further use. Of all the isolates, 12 Alternaria and 19 Colletotrichum isolates representative of the entire variability, were used for further characterization (Supplementary Table S1).

Morphological Characterization
Representative fungal isolates of Alternaria and Colletotrichum were grown on PDA amended with streptomycin sulphate (100 mg/L) at 25 ± 1 • C. Seven days after incubation, all the isolates were subjected to macroscopic and microscopic study by using a compound microscope (Nikon Eclipse 90i). Growth rate, colony appearance and conidium characteristics were recorded for each isolate of Alternaria and Colletotrichum according to the method previously described [21,37]. Statistical analyses of the data obtained were performed using WASP (Web Agri Stats Package) software available at https://ccari.icar.gov.in/waspnew.html (accessed on 30 June 2022). Multivariate statistical analysis, such as Principal Component Analysis, was performed using morphological data such as length and width of conidia.

Molecular Characterization
A multi-locus approach was employed to characterize the selected isolates of Alternaria and Colletotrichum. The barcoding genetic regions, such as ITS, LSU, NS and TEF-1 for Alternaria and ITS, GADPH and ACT for Colletotrichum, were PCR amplified and sequenced for characterization (Table 1). For genomic DNA isolation, mycelium was harvested from colonies of fungal isolates grown on PDA after 7 days of incubation at 24 ± 1 • C. Genomic DNA was extracted using the CTAB method described earlier [38] with some modifications. PCR was carried out in a 10 µL reaction mixture containing 1 µL of 50 ng g DNA, 0.25 µL of 10 µM primer forward and reverse each (Table 1), 4 µL of 2X PCR master mix (HiMedia Laboratories Pvt Ltd., Mumbai, India) and 4.5 µL molecular-grade sterile water. PCR was performed in a Thermocycler (HiMedia, Laboratories Pvt Ltd., Mumbai, India) with the following PCR program for ITS, LSU, NS and TEF-α amplification: initial denaturation at 95 • C for 3 min, followed by 35 cycles of denaturation (95 • C for 30 s), annealing (55 • C for 30 s), extension (72 • C for 30 s) and the final extension at 72 • C for 7 min. Amplification of GAPDH and ACT was performed using touchdown PCR with an initial denaturation at 94 • C for 5 min, followed by 16 cycles of denaturation (94 • C for 30 s), annealing (60 • C for 30 s) and extension (72 • C for 45 s), followed by 25 cycles of denaturation (94 • C for 30 s), annealing (55 • C for 30 s), extension (72 • C for 45 s) and the final extension at 72 • C for 10 min. The PCR product was resolved on 1% agarose gel and sequenced using Sanger sequencing at a commercial facility (Eurofins Genomics India Pvt. Ltd., Bengaluru, India). The obtained sequences were screened using Finch TV v 1.4.0 and searched against NCBI database using homology search (BLASTn). After validation, consensus sequences were deposited at GenBank, an NCBI database, with accession numbers given in Tables 2 and 3.

Phylogenetic Analysis
Phylogenetic analysis was performed using sequences obtained from the PCR amplification of genetic regions of the Alternaria (ITS, LSU, NS and TEF-α) and Colletotrichum (TUB, CHS, ITS, GADPH and ACT) isolates used in the current study and validated representative sequences of different Alternaria and Colletotrichum species available in the database. Individual sequences were aligned using the MUSCLE algorithm in MEGA XI software [44] and a phylogenetic tree was constructed using the Maximum Likelihood method and the Tamura-Nei model; analysis was performed with 1000 bootstrap replications. Aligned sequences were also concatenated to obtain multi-locus sequences and used for phylogenetic tree construction using the Maximum Likelihood method and the Tamura-Nei model. Multi-locus sequence analysis (MLSA) was performed with 1000 bootstrap replications. A combined dataset of coding and non-coding regions was used in order to maximize the effectiveness of the genetic diversity analysis amongst Alternaria and Colletotrichum isolates obtained in the current study [19].

Pathogenicity tests
To prove Koch's postulates, pathogenicity tests for selected isolates of Alternaria and Colletotrichum were performed in vitro using the mycelial plug inoculation method with some modifications [27]. Briefly, fresh healthy fruits of cv. Bhagawa were collected from bearing orchards (rainy season crop) of the National Research Centre (ICAR) on Pomegranate, Solapur, Maharashtra. Fruits were collected from the orchard where no pesticide sprays were used for the last 25 days and washed with distilled water to remove surface contaminants. Fruits were further disinfected with 70% ethanol and placed on plastic mesh platforms inside sterile glass jar chambers (diameter × height: 30 cm × 22.5 cm). Sterile distilled water was added underneath the platform (12-15 cm) to maintain high humidity (>70 %). Five fruits were inoculated (per isolate) with mycelial plugs (4 mm) at wounded and non-wounded sites from seven-day-old cultures grown on PDA. Healthy fruits were inoculated with PDA alone, which served as the control (Supplementary Figure S1). The inoculated fruits were incubated at room temperature for 7 days and observed regularly for onset of the symptoms.

Symptoms and Disease Incidence
Fungal pathogens belonging to the genus Alternaria and Colletotrichum cause disease on several horticultural plant species [28,[45][46][47][48]. They have been reported to be destructive pathogens infecting pomegranate worldwide [6,25,28,49,50]. Alternaria causes heart rot of fruits and leafspot/blight, while Colletotrichum causes fruit anthracnose. In the current study, fruits with natural infections of Alternaria collected in various regions of India did not exhibit any damage or signs of rotting on the outer surface of the peel; however, they exhibited a peculiar external coloration of the peel (Figure 1a). When such fruits were cut open, the arils inside were brown and rotten ( Figure 1b). The infected fruits at advanced stage produced a hollow sound when knocked, while healthy fruits did not. Moreover, infected fruits were lighter than healthy fruits of comparable size and age. These symptoms are characteristic of heart rot of pomegranate [25], and as such it may be difficult to identify heart rot visibly externally. Fruits infected with Colletotrichum exhibited characteristic brown-tan hard spots on the surface (Figure 1c). The lesions often displayed gray-orange fungal spore masses on the surface and expanded into the rind and arils, leading to fruit decay [51].
As per yearly data recorded in field surveys at the National Research Centre (ICAR) from 2015 to 2019, the incidence of pomegranate fruit rot ranged from 0 to 8% in the case of Alternaria and 0-27% in the case of Colletotrichum. However, a remarkable increase was observed in disease incidence in the last two years (2020-2022): up to 25% for Alternaria rot and 63% for Colletotrichum rot. During these surveys, infected samples were collected from pomegranate orchards in different geographical locations in India, from which around 30 and 45 isolates of Alternaria and Colletotrichum, respectively, were recovered. Out of these isolates, 12 Alternaria and 19 Colletotrichum isolates representing different geographical regions were selected based on their morphotypes (Table 2 and Supplementary Table S1). Most of the selected isolates were from Maharashtra, the leading area for pomegranate production and export. did not exhibit any damage or signs of rotting on the outer surface of the peel; however, they exhibited a peculiar external coloration of the peel (Figure 1a). When such fruits were cut open, the arils inside were brown and rotten ( Figure 1b). The infected fruits at advanced stage produced a hollow sound when knocked, while healthy fruits did not. Moreover, infected fruits were lighter than healthy fruits of comparable size and age. These symptoms are characteristic of heart rot of pomegranate [25], and as such it may be difficult to identify heart rot visibly externally. Fruits infected with Colletotrichum exhibited characteristic brown-tan hard spots on the surface (Figure 1c). The lesions often displayed gray-orange fungal spore masses on the surface and expanded into the rind and arils, leading to fruit decay [51]. As per yearly data recorded in field surveys at the National Research Centre (ICAR) from 2015 to 2019, the incidence of pomegranate fruit rot ranged from 0 to 8% in the case of Alternaria and 0-27% in the case of Colletotrichum. However, a remarkable increase was observed in disease incidence in the last two years (2020-2022): up to 25% for Alternaria rot and 63% for Colletotrichum rot. During these surveys, infected samples were collected from pomegranate orchards in different geographical locations in India, from which around 30 and 45 isolates of Alternaria and Colletotrichum, respectively, were recovered. Out of these isolates, 12 Alternaria and 19 Colletotrichum isolates representing different geographical regions were selected based on their morphotypes (Table 2 and Supplementary  Table S1). Most of the selected isolates were from Maharashtra, the leading area for pomegranate production and export.

Morphological Characterization of Isolates
Colonies produced by Alternaria and Colletotrichum isolates, when cultured on PDA, displayed intra-genus variability of morphological features. Alternaria isolates could be broadly grouped into three morphotypes based on colony morphology ( Figure 2). Morphotype I: colonies that appeared greyish white on the obverse; on the reverse, the innermost part was dark brown with brownish white margins. Eight isolates (Alt-1, 3, 5, 6, 7, 8, JD and N) belonged to this morphotype. Morphotype II: colonies that appeared white on the obverse and light-brown with a white margin on the reverse. Isolates Alt-12 and 13 belonged to this morphotype. Morphotype III: colonies that appeared brownish on the obverse and light-brown with a white margin on the reverse. Isolates Alt-2 and MP6 belonged to this morphotype. The color of colonies (grey-white-brown) produced by Alternaria isolates in the current study (Supplementary Table S2) are characteristic of this genus [52,53]. The growth rate of different isolates ranged from 4.5 to 7 mm per day, with a maximum exhibited by isolate Alt-8 and a minimum by isolate Alt-13 (Table 4)

Morphological Characterization of Isolates
Colonies produced by Alternaria and Colletotrichum isolates, when cultured on PDA, displayed intra-genus variability of morphological features. Alternaria isolates could be broadly grouped into three morphotypes based on colony morphology ( Figure 2). Morphotype I: colonies that appeared greyish white on the obverse; on the reverse, the innermost part was dark brown with brownish white margins. Eight isolates (Alt-1, 3, 5, 6, 7, 8, JD and N) belonged to this morphotype. Morphotype II: colonies that appeared white on the obverse and light-brown with a white margin on the reverse. Isolates Alt-12 and 13 belonged to this morphotype. Morphotype III: colonies that appeared brownish on the obverse and light-brown with a white margin on the reverse. Isolates Alt-2 and MP6 belonged to this morphotype. The color of colonies (grey-white-brown) produced by Alternaria isolates in the current study (Supplementary Table S2) are characteristic of this genus [52,53]. The growth rate of different isolates ranged from 4.5 to 7 mm per day, with a maximum exhibited by isolate Alt-8 and a minimum by isolate Alt-13 (Table 4). Conversely, Colletotrichum isolates could be broadly grouped into two morphotypes ( Figure 3). Morphotype I comprised isolates that produced colonies which appeared white and fluffy (Col-1, 4, 11, 18, and 21), mostly with regular margins on the obverse and paleyellow to yellow on the reverse. The majority of isolates (Col-2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 25, 26 and 27) belonged to morphotype II, which produced colonies that appeared grey in the middle with whitish irregular margins on the obverse and greyish yellow on the reverse. Such morphological features of colonies produced by Colletotrichum isolates in the current study (Supplementary Table S3) are characteristic of Colletotrichum as reported in previous studies by other authors [27,51]. The growth rate of different isolates ranged from 3.29 to 11.14 mm per day (Table 4). Conversely, Colletotrichum isolates could be broadly grouped into two morphotypes ( Figure 3). Morphotype I comprised isolates that produced colonies which appeared white and fluffy (Col-1, 4, 11, 18, and 21), mostly with regular margins on the obverse and paleyellow to yellow on the reverse. The majority of isolates (Col-2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 25, 26 and 27) belonged to morphotype II, which produced colonies that appeared grey in the middle with whitish irregular margins on the obverse and greyish yellow on the reverse. Such morphological features of colonies produced by Colletotrichum isolates in the current study (Supplementary Table S3) are characteristic of Colletotrichum as reported in previous studies by other authors [27,51]. The growth rate of different isolates ranged from 3.29 to 11.14 mm per day (Table 4).  Conversely, Colletotrichum isolates could be broadly grouped into two morphotypes ( Figure 3). Morphotype I comprised isolates that produced colonies which appeared white and fluffy (Col-1, 4, 11, 18, and 21), mostly with regular margins on the obverse and paleyellow to yellow on the reverse. The majority of isolates (Col-2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 25, 26 and 27) belonged to morphotype II, which produced colonies that appeared grey in the middle with whitish irregular margins on the obverse and greyish yellow on the reverse. Such morphological features of colonies produced by Colletotrichum isolates in the current study (Supplementary Table S3) are characteristic of Colletotrichum as reported in previous studies by other authors [27,51]. The growth rate of different isolates ranged from 3.29 to 11.14 mm per day (Table 4).

Morphotype-I
Morphotype-II   Conidia produced by the majority of Alternaria isolates were ovoid, with only two isolates (Alt-8 and Alt-13) producing obclavate conidia, and appeared light-to-dark-brown under the microscope. Conidial length ranged from 11 to 27 µm, width from 5 to 8 µm, while the beak length varied from 1.9 to 3.7 µm (Supplementary Table S4). Conidia produced by Alt-2 were the shortest and those by Alt-8 were the longest (Figure 4). However, the beak length was shortest in the Alt-7 isolate and longest in the Alt-JD isolate. The dimensions of conidia, as observed in the current study, corresponded to those reported previously for small spore species of Alternaria [54]. The number of horizontal septa ranged from 2 to 5, while the number of vertical septa ranged from 0 to 2 amongst the isolates characterized in this study (Supplementary Table S4).
Conidia produced by the majority of Alternaria isolates were ovoid, with only two isolates (Alt-8 and Alt-13) producing obclavate conidia, and appeared light-to-darkbrown under the microscope. Conidial length ranged from 11 to 27 μm, width from 5 to 8 μm, while the beak length varied from 1.9 to 3.7 μm (Supplementary Table S4). Conidia produced by Alt-2 were the shortest and those by Alt-8 were the longest (Figure 4). However, the beak length was shortest in the Alt-7 isolate and longest in the Alt-JD isolate. The dimensions of conidia, as observed in the current study, corresponded to those reported previously for small spore species of Alternaria [54]. The number of horizontal septa ranged from 2 to 5, while the number of vertical septa ranged from 0 to 2 amongst the isolates characterized in this study (Supplementary Table S4). Colletotrichum isolates produced cylindrical, hyaline, single-celled conidia. Conidia of isolates Col-1, 2, 3, 9, 11, 12, 13, 14, 15, 17, 18 and 25 were rounded at both ends, while conidia of the rest of the isolates were pointed at one end and rounded at the other. Conidial length ranged from 7.9 to 17 μm and width from 2.1 to 3.2 μm, with a length-towidth ratio ranging between 2.7 and 5.3 ( Figure 5, Supplementary Table S5). Colletotrichum isolates produced cylindrical, hyaline, single-celled conidia. Conidia of isolates Col-1, 2, 3, 9, 11, 12, 13, 14, 15, 17, 18 and 25 were rounded at both ends, while conidia of the rest of the isolates were pointed at one end and rounded at the other. Conidial length ranged from 7.9 to 17 µm and width from 2.1 to 3.2 µm, with a length-to-width ratio ranging between 2.7 and 5.3 ( Figure 5, Supplementary Table S5). Principal component analysis (PCA) was performed using conidial features for isolates of both Alternaria and Colletotrichum. However, based on these characteristics, no clustering or grouping could be obtained (Figure 6), confirming that morphological features alone are not sufficient to separate and identify the isolates [55]. Delimiting species boundaries and accurate identification of species belonging to the Alternaria genus within the Alternaria section is difficult because of the overlapping of characteristics and morphological plasticity under different cultural conditions [56]. Therefore, morphological characterization accompanied by molecular information has been utilized for species identification in Alternaria species [57]. Similarly, for Colletotrichum, the morphological features are not reliable for identifying species and defining species boundaries. Therefore, other features based on DNA/RNA sequences, secondary metabolite production or pathogenicity have been employed either alone or in combination for accurate identification of Colletotrichum species [16]. Principal component analysis (PCA) was performed using conidial features for isolates of both Alternaria and Colletotrichum. However, based on these characteristics, no clustering or grouping could be obtained (Figure 6), confirming that morphological features alone are not sufficient to separate and identify the isolates [55]. Delimiting species boundaries and accurate identification of species belonging to the Alternaria genus within the Alternaria section is difficult because of the overlapping of characteristics and morphological plasticity under different cultural conditions [56]. Therefore, morphological characterization accompanied by molecular information has been utilized for species identification in Alternaria species [57]. Similarly, for Colletotrichum, the morphological features are not reliable for identifying species and defining species boundaries. Therefore, other features based on DNA/RNA sequences, secondary metabolite production or pathogenicity have been employed either alone or in combination for accurate identification of Colletotrichum species [16].
logical plasticity under different cultural conditions [56]. Therefore, morphological characterization accompanied by molecular information has been utilized for species identification in Alternaria species [57]. Similarly, for Colletotrichum, the morphological features are not reliable for identifying species and defining species boundaries. Therefore, other features based on DNA/RNA sequences, secondary metabolite production or pathogenicity have been employed either alone or in combination for accurate identification of Colletotrichum species [16].

Molecular Characterization
Despite grouping into different morphotypes based on colony morphology, conidial features could not group the isolates. Since species belonging to Alternaria or Colletotrichum could not be adequately discriminated using morphological features alone, molecular approaches were also used. Individual genetic regions that were PCR amplified and sequenced were searched against the NCBI database. However, based on the BLASTn results, no single species could be deduced, and thus only genera were confirmed based on a homology search. Multi-locus phylogeny was applied for resolving such ambiguity at the species identification level. In particular, ITS, LSU, NS and TEF-α regions were targeted for identifying species of Alternaria, and ITS, ACT and GAPDH for Colletotrichum species (Table 1). Phylogenetic trees were drawn for individual fragments; however, the trees were not congruent ( Supplementary Figures S2-S7). To resolve the incongruence posed by single-gene phylogeny, a combined dataset of coding and non-coding regions was used in order to maximize the effectiveness of the genetic diversity analysis amongst Alternaria and Colletotrichum isolates obtained in the current study. Since NS is not able to provide much information useful for species identification [56], it was excluded from the combined dataset.

Phylogenetic Analyses
Based on the result of multi-locus phylogenetic analysis, Alternaria isolates clustered into two groups: group I, containing seven isolates (Alt-1, 3, 5, 6, 8, JD and N), showing close relatedness with Alternaria species belonging to the A. alternata species complex; and group II, containing five isolates (Alt-2, 7, 12, 13 and MP6), showing close relatedness to A. burnsii (Figure 7). All isolates of morphotype I, except Alt-7, were in group I, while isolates of morphotype II and III were in group II, indicating a partial overlap between colony morphology and multi-gene phylogeny. The presence of A. arborescens close to A. alternata in group I could be due to inconsistencies related to the A. arborescens species complex (AASC) reported in the literature [22,56,58,59]. For example, some studies have reported AASC to be distinct from the A. alternata species complex, while others have identified AASC as a subspecies of A. alternata or a different morphotype. However, A. arborescens has been retained in Alternaria sect. Alternaria [56]. Consistently, the isolates indicated under group I in the current study were referred to Alternaria sect. Alternaria. Moreover, a number of species of Alternaria, including A. solani, have been reported to infect pomegranate [22,28,60]; however, many of these species were not represented among the isolates characterized in the current study. Alternaria species, due to their high adaptivity to different environmental conditions, can infect pomegranate fruits in both pre-and post-harvest stages [61]; they produce toxins which are important for their virulence and may contaminate fruits and the products processed downstream [22]. Moreover, some species, such as A. gaisen, are quarantine pathogens imposing export restrictions, and therefore accurate identification of Alternaria species also has toxicological and phytosanitary implications. For multi-locus phylogeny of Colletotrichum isolates, sequences for species belonging to the Colletotrichum gloeosporioides species complex were retrieved [62]. In total, 52 species were included in the analysis, along with 17 isolates characterized in the current study. The combined dataset obtained by concatenation of five loci were used, and in case the sequence was not available, gaps were used in the alignment. As per the ML tree, the isolates belonging to morphotype I resolved well; for example, Col-1 and Col-4 showed close proximity to C. viniferum, Col-8 and Col-18 clustered together with Colletotrichum tainanense, while Col-21 grouped with Colletotrichum hederiicola. On the other hand, isolates belonging to morphotype II formed a separate cluster, showing closeness to the cluster containing Colletotrichum theobromicola and C. pseudotheobromicola. This cluster also contained Col-11, which belonged to morphotype I (Figure 8). Colletotrichum is one of the most economically important and highly damaging genus of plant pathogens and comprises several species complexes [62]. Colletotrichum gloeosporioides, long considered as a single species, is now regarded as one of the 14 Colletotrichum species complexes and encompasses 22 species [63][64][65][66][67]. In our study, some of the isolates showed close association with C. theobromicola, while others grouped with other species in the C. gloeosporioides species complex, indicating high genetic variability amongst them. Diversity of Colletotrichum species associated with the same host plant has also been reported for many anthracnose diseases of horticultural crops, such as citrus and olive, to name a few [15,[68][69][70][71]. For multi-locus phylogeny of Colletotrichum isolates, sequences for species belonging to the Colletotrichum gloeosporioides species complex were retrieved [62]. In total, 52 species were included in the analysis, along with 17 isolates characterized in the current study. The combined dataset obtained by concatenation of five loci were used, and in case the sequence was not available, gaps were used in the alignment. As per the ML tree, the isolates belonging to morphotype I resolved well; for example, Col-1 and Col-4 showed close proximity to C. viniferum, Col-8 and Col-18 clustered together with Colletotrichum tainanense, while Col-21 grouped with Colletotrichum hederiicola. On the other hand, isolates belonging to morphotype II formed a separate cluster, showing closeness to the cluster containing Colletotrichum theobromicola and C. pseudotheobromicola. This cluster also contained Col-11, which belonged to morphotype I (Figure 8). Colletotrichum is one of the most economically important and highly damaging genus of plant pathogens and comprises several species complexes [62]. Colletotrichum gloeosporioides, long considered as a single species, is now regarded as one of the 14 Colletotrichum species complexes and encompasses 22 species [63][64][65][66][67]. In our study, some of the isolates showed close association with C. theobromicola, while others grouped with other species in the C. gloeosporioides species complex, indicating high genetic variability amongst them. Diversity of Colletotrichum species associated with the same host plant has also been reported for many anthracnose diseases of horticultural crops, such as citrus and olive, to name a few [15,[68][69][70][71].   Figure 8. Multi-locus sequence analysis based on five genomic regions (TUB, CHS, ITS, ACT and GAPDH) amplified from different isolates of Colletotrichum obtained from pomegranate in India and reference isolates of diverse species belonging to the Colletotrichum gloeosporioides species complex. The Maximum Likelihood (ML) tree was drawn using MEGA XI with 1000 bootstraps. Isolates in red belong to morphotype I, while those in blue to morphotype II.
Since different Alternaria and Colletotrichum species have different sensitivity toward the commercially available and routinely applied fungicides [10][11][12]72,73], the complete and precise knowledge of the species involved in the etiology of heart rot and anthracnose of pomegranate fruits is the first step toward devising effective management strategies aimed at preventing these two major fungal diseases of pomegranate in India.

Pathogenicity Tests
Representative isolates were tested for their pathogenicity on detached fruits. Alternaria isolates obtained from both fruits and leaves induced rotting on the surface of fruit peel, as well as inside the fruit in the arils, 12 days after inoculation (Supplementary Figure S1). Alternaria alternata isolates produced both types of symptoms, while other species of Alternaria caused internal rotting only. All Colletotrichum isolates induced characteristic symptoms of anthracnose 12-15 days after inoculation (Supplementary Figure S1). Thus, Koch's postulates were verified for both the pathogens.

Conclusions
The present study highlighted the variability of Alternaria and Colletotrichum associated with pomegranate in India and showed that heart rot and anthracnose of pomegranate fruits are caused by diverse Alternaria and Colletotrichum species, respectively. Most of these species were reported previously in other pomegranate-growing countries. However, many of the taxa identified in this study are first records on pomegranate in India, and A. burnsii is reported for the first time as a pathogen of pomegranate worldwide.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8101040/s1, Figure S1: Flowchart depicting steps performed during pathogenicity tests. Figure S2: Phylogenetic analysis using elongation factor (TEF-α) gene amplified and sequenced from Alternaria isolates. Maximum Likelihood tree drawn using MEGA XI with 1000 bootstraps. Figure S3: Phylogenetic analysis using ITS region amplified and sequenced from Alternaria isolates. Maximum Likelihood tree drawn using MEGA XI with 1000 bootstraps. Figure S4: Phylogenetic analysis using nuclear ribosomal large subunit (LSU) region amplified and sequenced from Alternaria isolates. Maximum Likelihood tree drawn using MEGA XI with 1000 bootstraps. Figure S5: Phylogenetic analysis using actin gene amplified and sequenced from Colletotrichum isolates. Maximum Likelihood tree drawn using MEGA XI with 1000 bootstraps. Figure S6: Phylogenetic analysis using GAPDH (glyceraldehyde phosphate dehydrogenase) gene amplified and sequenced from Colletotrichum isolates. Maximum Likelihood tree drawn using MEGA XI with 1000 bootstraps. Figure S7: Phylogenetic analysis using ITS region amplified and sequenced from Colletotrichum isolates. Maximum Likelihood tree drawn using MEGA XI with 1000 bootstraps. Table S1: Origin of isolates of Alternaria (n = 12) and Colletotrichum (n = 19) from pomegranate characterized in this study. Table S2: Morphotypes of Alternaria isolates (n = 12) characterized in the current study. Front and back of seven-day-old colonies grown on PDA at 25 • C. Table S3: Morphotypes of Colletotrichum isolates (n = 19) characterized in the current study. Front and back of seven-day-old colonies grown on PDA at 25 • C. Table S4: Microscopic features of conidia produced by different isolates (n = 12) of Alternaria characterized in this study.