RESUMO
In March 2022, cankers and lesions appeared on the branches of 2-3-year-old pomegranate plants grown in four orchards of Hanumangarh, Rajasthan, India. The disease incidence ranged from 5-15%. Field symptoms such as dark brown lesions on one side of the branches, cracked lesions, vascular tissue discoloration and drooping of the plants were noticed. To identify the causative agent, 2 diseased branch samples, showing typical symptoms collected from each orchard 25-30 km apart. The samples were washed with distilled water and small sections of tissue were excised from both symptomatic and asymptomatic areas using a sterile scalpel. Sections were surface sterilized with 1% sodium hypochlorite for 30 sec and 70% ethanol for 2 min followed by rinsing with sterilized water thrice. Sterile sections were dried on sterile filter paper and then transferred onto potato dextrose agar (PDA) amended with streptomycin (100 mgL-1) and incubated at 24±1°C in the dark. Samples (n=5) collected from different orchards produced similar colonies, with greyish white aerial mycelia, which became dark black after 5-7 days. The morphological characteristics of all isolates were observed under microscope. Immature conidia (6.3±1.05*14.7±0.98 µm: average of 50 measurements) were single celled, hyaline, ellipsoid or ovoid, apex rounded and truncated at the base while the matured conidia (8.4±1.41*15.3±1.17 µm: average of 50 measurements) had two cells with dark septa. The conidial morphology of all isolates was in accordance with Lasiodiplodia sp. (Alves et al; 2008) therefore, one representative isolate (HSC-1) was used for molecular identification at species level. Three loci viz., ITS, EF1-a and ß tubulin of fungal genomic DNA were PCR amplified using ITS-1/4, EF-F/R and TUB-2A/2B primers, respectively. The amplicons were sequenced and deposited in GenBank, NCBI database with accession no. ON598885 (ITS), ON605203 (EF) and ON605204 (TUB). BLASTn analysis showed similarity with the sequences of Lasiodiplodia theobromae isolates: ITS showed 100% with MK530071.1 (492 bases), EF 99.77% with MT975688.1 (436 bases) and BT 99.76% with MW287586.1 (422 bases). Phylogenetic analysis using Neighbour Joining method revealed close association among L. theobromae isolates. Thus, causative agent associated with stem canker of pomegranate was confirmed as L. theobromae. Further, the same isolate was used for pathogenicity tests on 1-year-old pomegranate plants (n=6). Briefly, 2 cm wound was created in the main stem with a sterile scalpel and a same-size mycelial plug was placed in the wound and wrapped with parafilm. Six plants that were wrapped with uncultured PDA served as control. The inoculated plants were maintained at 26°C and 65-70% RH in a polyhouse. After 4 days parafilm was removed from all plants. The experiment was repeated twice. Inoculated plants produced lesions (0.7 x 5.5 cm; average of 6 measurements) similar to field symptoms after 10-15 days and no such symptoms developed on control plants. The difference between control and inoculated plants was statistically significant (p=0.0001). The fungus was re-isolated from symptomatic tissue and colonies were morphologically similar to HSC-1, thus fulfilling the Koch's postulates. The fungus, L. theobromae causes stem canker and dieback on different host plants and is mainly distributed in tropical and subtropical regions and has been reported on pomegranate from Florida (Xavier et al 2017). To the best of our knowledge, this is the first report of L. theobromae causing stem canker of pomegranate in India.
RESUMO
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.
RESUMO
Despite the availability of whole genome assemblies, the identification and utilization of gene-based marker systems has been limited in pomegranate. In the present study, we performed a genome-wide survey of intron length (IL) markers in the 36,524 annotated genes of the Tunisia genome. We identified and designed a total of 8,812 potential intron polymorphism (PIP) markers specific to 3,445 (13.40%) gene models that span 8 Tunisia chromosomes. The ePCR validation of all these PIP markers on the Tunisia genome revealed single-locus amplification for 1,233 (14%) markers corresponding to 958 (27.80%) genes. The markers yielding single amplicons were then mapped onto Tunisia chromosomes to develop a saturated linkage map. The functional categorization of 958 genes revealed them to be a part of the nucleus and the cytoplasm having protein binding and catalytic activity, and these genes are mainly involved in the metabolic process, including photosynthesis. Further, through ePCR, 1,233 PIP markers were assayed on multiple genomes, which resulted in the identification of 886 polymorphic markers with an average PIC value of 0.62. In silico comparative mapping based on physically mapped PIP markers indicates a higher synteny of Tunisia with the Dabenzi and Taishanhong genomes (>98%) in comparison with the AG2017 genome (95%). We then performed experimental validation of a subset of 100 PIP primers on eight pomegranate genotypes and identified 76 polymorphic markers, with 15 having PIC values ≥0.50. We demonstrated the potential utility of the developed markers by analyzing the genetic diversity of 31 pomegranate genotypes using 24 PIP markers. This study reports for the first time large-scale development of gene-based and chromosome-specific PIP markers, which would serve as a rich marker resource for genetic variation studies, functional gene discovery, and genomics-assisted breeding of pomegranate.
RESUMO
Present research discovered novel miRNA-SSRs for seed type trait from 761 potential precursor miRNA sequences of pomegranate. SSR mining and BLASTx of the unique sequences identified 69 non-coding pre-miRNA sequences, which were then searched for BLASTn homology against Dabenzi genome. Sixty three true pri-miRNA contigs encoding 213 pre-miRNAs were predicted. Analysis of the resulting sequences enabled discovery of SSRs within pri-miRNA (227) and pre-miRNA sequences (79). A total of 132 miRNA-SSRs were developed for seed type trait from 63 true pri-miRNAs, of which 46 were specific to pre-miRNAs. Through ePCR, 123 primers were validated and mapped on eight Tunisia chromosomes. Further, 80 SSRs producing specific amplicons were ePCR-confirmed on multiple genomes i.e. Dabenzi, Taishanhong, AG2017 and Tunisia, yielding a set of 63 polymorphic SSRs (polymorphism information content ≥0.5). Of these, 32 miRNA-SSRs revealed higher polymorphism level (89.29%) when assayed on six pomegranate genotypes. Furthermore, target prediction and network analysis suggested a possible association of miRNA-SSRs i.e. miRNA_SH_SSR69, miRNA_SH_SSR36, miRNA_SH_SSR103, miRNA_SH_SSR35 and miRNA_SH_SSR53 with seed type trait. These miRNA-SSRs would serve as important genomic resource for rapid and targeted improvement of seed type trait of pomegranate.
