Genetic diversity of Prosopis juliflora in the state of Qatar and its valuable use against postharvest pathogen of mango fruits

Prosopis juliflora leaves collection and processing for Ribotyping

Prosopis juliflora species of the genus Prosopis, family of Fabaceae had its genetic variation in Doha evaluated. Seven samples of P. juliflora leaves were collected from six different locations in Doha, Qatar, during five field trips. Plant leaves were collected after proper permissions and all methods were carried out in accordance with relevant guidelines and regulations. Trees in all locations were naturally growing around urbanization areas in their normal arid habitat without artificial irrigation, samples were collected from fully mature trees. Table 1 shows the samples details. Figure 1 shows the location sites of where the samples were collected on the map of Qatar, Doha. Leaf samples were kept in sterile labeled bags until having reached the laboratory where few leaflets were washed with sterile distilled water and sterilized using 70% ethanol to be used for DNA extraction.

Ribotyping analysis

The leaflets of each sample were transferred into a sterile mortar previously cooled at -20 ˚C and used for DNA extraction following the kit manufacturer instructions (DNeasy Plant Mini Kit-QIAGEN-USA).

Extracted DNA of each sample were subject to PCR using ITS1 and ITS4 primers. PCR products obtained were purified using the Invitrogen Quick PCR Purification Kit (QIAGEN, Germany) as indicated by the manufacturer and sequenced using Sanger sequencer (3130/3130xl DNA Analyzers, Thermofisher Scientific, USA) as previously described²².

Sanger sequencer raw data was read using BioEdit software. Basic Local Alignment Search Tool (BLAST) network services of the National Centre for Biotechnology Information (NCBI) database were used to compare the obtained sequences to the existing sequences. Sequences were submitted to NCBI for accession numbers. The various P. juliflora ribosomal sequences obtained were also uploaded on MEGA-X software and the phylogeny tree was generated using the neighbor-joining algorithm²⁶.

Minimum inhibitory concentrations of PJ-WS-LE extracts prepared using leaf samples collected from various locations against A. alternata and C. gloeosporioides

Preparation of PJ-WS-LE extract

Fresh, young full leaves of P. juliflora were collected from various locations as indicated in Fig. 1. Samples were washed, dried and ground into powder to be used in the preparation of PJ-WS-LE extract as previously described²². Briefly, every 20 g of the leaf powder were incubated in 200 mL of 70% ethanol for 48 h. The supernatant has its solvent evaporated, the extract was then re-dissolved in sterile distilled water. Only water-soluble phytochemicals were tested by centrifuging the final preparation tubes and excluding the pellet. Stock solution of 100 g L⁻¹ was stored at 4 °C to be used for later experiments. PJ-WS-LE extract concentration used in treatments was 8 g L⁻¹ which is double the highest minimum inhibitory concentration of the extract against spoiling microorganisms as previously determined²².

Determination of minimum inhibitory concentration

The MIC test was conducted in a sterile 96-well plate, with each well containing 100 μl of potato dextrose broth (PDB) (HIMEDIA-India). Every four wells made one replication, nine different concentrations of the crude extracts were tested (1:1 dilutions) ranging from 42 to 0.16 g L⁻¹. Wells were then inoculated with one of the two tested microorganisms’ spore suspensions (A. alternata and C. gloeosporioides). The last three rows are control rows: no spores and no extract control wells, negative control with spores but no extract wells, and positive control with spores and 10 µl of the fungicidal Clatrimazole (1%) wells.

Fungal spore suspensions were adjusted to the range of 10⁴ spores L⁻¹ using a 10 day old fungal plate and sterile distilled water, the spore concentration was calculated using a heamatocytometer.

Fungal growth in each well was monitored using Resazurin (HIMEDIA-India) dye. Upon cells division, Resazurin changes its color from blue to pink and fluorescent²⁷. Results were taken within 48 h of incubation at 25 °C. MIC was recorded as the last extract concentration that shows no change in the color of Resazurin within the incubation period.

Curative and preventive effects of PJ-WS-LE extract against A. alternata and C. gloeosporioides induced infection in mangoes

Pathogens

The two fungal strains used C. gloeosporioides and A. alternata were obtained from our laboratory collection, Department of Biological and Environmental Sciences, Qatar University, Qatar. Both fungal isolates were previously isolated from locally collected fruit samples. Isolates were molecularly identified by sequencing the Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) that was amplified by PCR. Identified fungal isolates were given the strains code of AaltQU17 for A. alternata and CgloQU17 for C. gloeosporioides²². Preserved cultures were sub-cultured on potato dextrose agar (PDA) plates and incubated at 25 °C for 10 days. Plates were then flooded with 10 mL of sterile distilled water each, to prepare the needed spores suspension solutions. The concentrations of spores suspensions were adjusted to 10⁶ spores L⁻¹ using a heamatocytometer¹⁸.

Fruit

The mango (Mangifera indica) type known as Neelam imported from India was used in the experiments. Fruit were bought from the whole sale market upon their arrival to the country. Only undamaged mature fruit were used in the experiment. Fruits chosen were ripen but not yet soft with firmness average of 20 ± 5.1 N, weight average of 177.61 ± 0.2 g and TSS average of 70 ± 5.3%. Fruit were first washed with sink water and sterilized twice with 70% ethanol to be then washed with sterile distilled water and left to air dry.

Preventive and curative effects of PJ-WS-LE extract

Wounded mango fruit were used during the experiment, the wounds were made through three needle pricks (2 mm deep) in three different places for each plant using a sterile syringe. A completely randomized design was used and each treatment was made of a triplicate of 10 fruit each. The experiment was repeated twice.

PJ-WS-LE extract of leaves collected from Qatar university field was first tested for its efficacy in preventing fungal contamination in wounded mango fruit (preventive effect). Therefore, the wounded zone of each fruit was sprayed with 8 g L⁻¹ PJ-WS-LE extract and then left to air-dry. Once dry the fruit were sprayed again with the extract at the same concentration and left to dry. Control fruit were only treated with sterile distilled water without the plants extract. After two hours all wounds were inoculated with 20 μL of conidia aqueous solution (10⁶ spores mL⁻¹) of one of the tested fungi. The extract was then tested for its ability to cure fungal contamination in wounded fruit. Therefore, wounds were inoculated first with 20 μL of conidia aqueous solution (10⁶ spores mL⁻¹) and left to dry. Wounds were then sprayed twice with 8 g L⁻¹ PJ-WS-LE extract.

All mangoes were stored in sterilized plastic trays inside an incubator at 25 °C and 75% humidity. Fruit were observed every 24 h for 5 days for C. gloeosporioides inoculated fruit and for 10 days for A. alternata inoculated fruit. Three parameters were recorded at the end of the experiment: disease incidence (DI), disease severity (DS), and percent plant extract efficacy (%EE). To calculate disease severity, the diameter of the infected area of each fruit was measured in two perpendicular directions and mean diameter mycelial growth was calculated^28,29.

End of the trial samples firmness

At the end of the trial, remaining mango fruit were tested for their flesh quality using a penetrometer (Agriculture Solutions, USA) to test the flesh firmness. Fruit were peeled, then the stainless steel probe of the instrument was inserted in three different points towards the equator of the fruit. Firmness in Newton was recorded and compared with standard fruit firmness to judge fruit quality¹⁸.

Effectiveness of PJ-WS-LE extract as long-term coating material and the preservative value of its chitosan-embedded form

Coating solutions preparation

Chitosan solution of 1% concentration was prepared by stirring chitosan powder (CAS 9012-76-4, Himedia, India) in 1% glacial acetic acid (IsoLab, Germany) overnight. The final chitosan solution pH was adjusted to 5.6 using 0.1 M NAOH (Sigma-Aldrich, Germany). To prepare PJ-WS-LE extract chitosan-embedded coating material, filter-sterilized PJ-WS-LE extract stock solution was added to 1% chitosan to achieve a final concentration of 8 g L⁻¹³⁰.

Samples preparation

Eighty-four mango samples chosen as described above, were divided into four groups of 18 samples each. Samples were divided into four treatment batches and treated as following:

• Batch A: non-treated fruit.

• Batch B: PJ-WS-LE extract at 8 g L^–1 was used to spray the fruit.

• Batch C: 1% chitosan was used to spray the fruit.

• Batch D: 8 g L⁻¹ PJ-WS-LE extract embedded in 1% chitosan was used to spray the fruit.

Every experimental replicate was made up of three mango samples that were stored together in one sterile bag at 4 °C. The number of replications per treatment was six. The experiment was repeated twice³¹.

Evaluation of sensory quality

A five-points scale was used for the evaluation of the sensory quality of the samples for overall quality, smell, and color change. The attributes were evaluated weekly using the fruit of one experimental replicate. Scores were given using the following scale: 5 points indicate “extremely liked”, 4 points indicate “liked”, 3 points indicate “acceptable” 2 points indicate “disliked” and 1 point indicates “extremely disliked”. The weekly average score per batch was also calculated³².

Estimation of weight loss

Upon treatment at day zero, all mango samples were weighed and their weights were recorded as initial weights. Weights of all remaining samples were measured at the end of every week. The variation between the start weight and weekly weights is calculated as weekly weight loss. The average percent of weekly weight loss of each batch was calculated³².

Determination of samples firmness

The samples of each experimental replicate evaluated on a weekly basis had their firmness measured as previously described. The weekly average samples firmness (N) of every treatment batch was also calculated³³.

pH measurement

Mango fruit of each experimental replicate were blended weekly into juice, after filtration, a digital pH meter (Jenway, UK) was used to measure pH. The weekly average fruit pH of every treatment batch was also calculated. The pH meter was calibrated using a buffer solution of pH 7³⁴.

Total soluble solids (TSS) measurement

Total soluble solids of the prepared mango juice samples were measured in percent brix using a refractometer (ANTAHI, New Zealand). The weekly average fruit TSS (%) for each treatment batch was also calculated. The refractometers was calibrated using distilled water³⁵.

DPPH radical scavenging assay

A 1/10 mango juice dilution was prepared using sterile distilled water. 100 μL of each dilution was mixed with 1 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (100 mg L⁻¹) to be incubated in the dark at 37 °C for 45 min. After incubation, samples were centrifuged and the pellet was discarded. The intensity of the change in color of the supernatant was measured by spectrophotometry at 517 nm using methanol as a blank. 100 μL of methanol in 1 mL DPPH was used as the control for the experiment. Percent radical scavenging activity was calculated as per the below formula:

The weekly average % radical scavenging activity for each treatment batch was finally calculated³¹.

Statistical analysis

The experimental design used was Completely Randomized Design (CRD). One-way ANOVA followed by Tukey Post-Hoc test was used to evaluate the significance of the weekly percent change in weight among treatment batches at P ≤ 0.05. The significances of pH and TSS variation within different treatment batches were evaluated using One-way ANOVA test at P ≤ 0.05. Data was presented as average ± standard error of the Means (SEM). SPSS (Ver. 27, SPSS Inc. Chicago, USA) was used to perform the statistical analysis tests.

Source: Ecology - nature.com