First report on molecular identification of Fusarium species causing fruit rot of mandarin (Citrus reticulata) in Bangladesh

Fungi isolation from the infected fruits

Research location and samples collection. The research was conducted at Professor Joarder DNA and Chromosome Research Lab., Department of Genetic Engineering and Biotechnology, University of Rajshahi, Bangladesh during 2018 to 2019. The ten different rotten Citrus reticulata fruits (Figure 1A) were collected from fruit market in Rajshahi, Bangladesh. Out of 10 fruits, three showed symptoms of rot which were used for pathogen isolation.

Figure 1. Naturally infected postharvest mandarin fruit showing symptoms of Fusarium rot and morphological phenotypes (F1).

(A) Infected mandarin fruit; (B) fungus growth; (C) clamydospores, hyphae, appressoria; and (D) conidia under the microscope from seven-day-old culture at 35°C on potato dextrose agar.

Fungi isolation from the infected fruits. Collected fruits were cleaned under running tap water to remove foreign agents and kept in a Biosafety Cabinet (Esco, Singapore). Moreover, the fruits were disinfested with 1% sodium hypochlorite (NaOCl) for 30 seconds, followed by five rinses in autoclave distilled water. Disinfested tissue was excised and plated on potato dextrose agar (PDA) (Hi-Media, India) at 35°C in the dark for three days. The colonies showing typical morphological characteristics including, colony color, pigmentation, growth rate and size of macroconidia of Fusarium species were selected (Hafizi et al., 2013) and isolated using the single spore technique (Chowdhury et al., 2019). Isolated colonies were transferred onto a Petri plate with PDA and incubated for seven days at 35°C in dark conditions. Isolates were grouped into two on the basis of morphological color (blackish color in the first group and whitish in the second group). Finally, one isolate from each of the two groups was selected for morphological and molecular analysis.

Morphological characterization of isolates

The selected fungal colony was characterized by macromorphological and micromorphological investigation (Al-Najada & Gherbawy, 2015). The isolate was sub-cultured in fresh PDA medium and three-day-old cultures were mounted using the lacto-phenol cotton blue (LPCB) staining method (Sathya et al., 2017). The mounted microscope slide was covered with a cover slip and conidia were observed under a light microscope (LABOMED LX400, USA) at 40X magnification.

Molecular characterization of Fusarium species

Genomic DNA was extracted from 15 gm of mycelia, collected from day three-day-old PDA cultures. DNA was extracted using a MaxMaxwell® 16 LEV Plant DNA Kit (Cat No. AS1420, Promega, USA) and DNA quantity and quality were checked using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA).

To amplify the internal transcribed spacer (ITS) gene, primer pairs ITS4 (5^′-TCCTCCGCTTATTGATATGC-3^′) and ITS5 (5^′-GGAAGTAAAAGTCGTAACAAGG-3^′) were used (Gardes & Bruns, 1993; White et al., 1990). The PCR reaction was performed using the method described by Hassan et al. (2018) using hot start green master mix (dNTPs, Buffer, MgCl2, Taq Pol) (Cat # M7432, Promega, USA). A total of 25 µl reaction volume containing 2 µl genomic DNA, 2.5 µl 1X PCR buffer, 1.0 µl MgCl₂, 1.5 µl dNTPs, 0.5 µl of each primer, 0.5 µl of Taq polymerase and 16.5 µl of deionized water was used. The PCR was programmed with an initialization step at 95°C for 2 min, followed by 32 cycles of denaturation at 95°C for 30 seconds, primer annealing at 48°C for 30 seconds, and extension at 72°C for 45 seconds and a final extension at 72°C for 10 minutes using a G02 GeneAtlas PCR machine (Astec, Japan). PCR products were run by horizontal electrophoresis (Mini-Gel, CBS Scientific, USA) on 1% agarose (Cat # V3125, Promega, USA) gel with 0.5% ethidium bromide solution (Cat # H5041, Promega, USA) in 1x TAE buffer (Cat # V4251, Promega, USA) using a 1kb DNA ladder (Cat # G5711, Promega, USA) as marker and visualized under alpha imager UV trans-illumination (Mini, Protein Simple, USA). PCR products were purified from the agarose gel, using the Wizard SV Gel and PCR Clean-Up System (Cat # A9281, Promega, USA).

Purified PCR products were sequenced commercially by Sanger sequencing (Apical Scientific, Malaysia). Sequences were used in a search using the NCBI BLAST tool. Sequences were submitted to GenBank and compared with the GenBank database. A phylogenetic tree was constructed using MEGA 10 software using the neighbor joining method (Kumar et al., 2016; Tamura et al., 2004).

Virulence test

Virulence competency of the isolate was carried out using the method described by Tafinta et al. (2013). The surfaces of mature, fresh guava, lemon and tomato were sterilized using water and 70% ethanol. The fruits were holed using a 2 mm sized cork borer and selected fungal inoculums were aseptically placed in the holes. The inoculated samples and the control were placed in sterile polythene bags and incubated at 35°C for seven days in dark. Isolates from the fruits and colonies from the diseased lesions were sub-cultured in PDA. The isolated fungal stains were identified based on colony features, growth rates and pigmentation. For confirmation, genomic DNA was isolated from subcultured colonies, which were isolated from artificially infected fruits. PCR amplification was performed using same procedure described in our previous article (Hasan et al., 2020) for virulence potency test through ITS rDNA gene amplification.

The study performed using a completely randomized design (CRD). Data were analyzed using Analysis of Variance (ANOVA) to see the differences between Fusarium species pathogenicity (Kasiamdari & Sangadah, 2015). The analysis was performed using SAS software version 9.4.

Morphological characterization

Collected samples were incubated on PDA medium following the single spore technique, and after seven days, white colored fungal colonies appeared (Figure 1B).

Isolated fungus was whitish in color and produced clamydospores, hyphae, appressoria and macroconidia (Figure 1C–D) at day three on PDA medium. Isolates produced microconidia that were 0-septate, oval, obovoid with a truncate base, elliptical or reniform. Macroconidia were sporodochia and fusiform. Chlamydospores were monophialides.

Molecular characterization

DNA amplification through PCR produced a bright band at approximately 650 bp where a 1kb DNA ladder was used as marker. No band was found in the negative control where water was used instead of template DNA (Figure 2).

Figure 2. PCR amplification of internal transcribed spacer region yielded ~650 bp product (F1).

1kb DNA marker is used for size determination, M - marker, I - isolate, N - negative control.

The dendrogram tree showed a close relationship with Fusarium concentricum and dissimilarity with Fusarium begoniae (Figure 3). Therefore, molecular identification confirmed the isolates as Fusarium sp. The sequence of the total isolate was compared to Fusarium sequences in GenBank using BLASTN, which revealed closely related sequences and 99.42% homology with the reference sequence for F. concentricum (Accession No. NR_111886.1).

Figure 3. Phylogenetic tree based on the internal transcribed spacer region of rRNA showing closest relatives of fungal species isolated from citrus fruit samples (F1).

The tree was constructed by neighbor joining method. The scale bar on the rooted tree indicates a 0.20 substitution per nucleotide position.

Virulence test

The virulence test was conducted to characterize the fungus as pathogenic or saprophytic on mature, fresh and healthy guava, lemon and tomato. All fruits showed similar morphological characteristics of Fusarium symptoms (Figure 4A–C). Isolated ribosomal DNA (rDNA) of fungus from artificially inoculated guava, lemon and tomato showed clear bands of approximately 650bp in length (Figure 5).

Figure 4. Fusarium rot symptoms in artificially inoculated fruits (F1).

(A) Guava, (B) lemon and (C) tomato, the image was taken 10 days after inoculation.

Figure 5. PCR amplification of internal transcribed spacer region yielded ~650 bp product for virulence test.

1kb DNA marker is used for size determination of inoculated fungus in different fruit samples. M - marker, G - guava, C - lemon, T - tomato, N - negative control.