Description and management of Aspergillus section Nigri causing post-harvest bulbs rot of onion

When onions are improperly stored, a post-harvest disease known as black mold of onion bulbs can result in considerable economic losses. Aspergillus section Nigri, one of many species, has been implicated in the development of black mold. In the present study, rot onion bulbs were collected from markets in Qena, Egypt. Thirteen Aspergillus section Nigri isolates were obtained and identified by morphological and molecular characterization. The ochratoxins potential of isolated A. section Nigri was tested, and three isolates were producers at the range of 1.5–15 ppm. For the presence of pks gene, no amplification product was detected. Using the fungal growth inhibition test, the isolates of A. niger were inhibited by eco-friendly materials Cement and Zeolite. Cement exhibited maximum percentage growth inhibition against the tested isolates at 74.7–86.7%. The pathogenicity activity of the A. niger isolates was tested by inoculation of healthy onion bulbs, other onion bulbs covered with Cement and Zeolite before inoculation by A. niger was used. The two treatments significantly reduced bulbs rot disease of onion than untreated bulbs. Seven and nine isolates showed 0% rot on covered bulbs by Cement and Zeolite, respectively as compared with inoculated onions, which exhibited rot ranging from 55 to 80%. Using eco-friendly materials with efficiency against post-harvest bulbs rot of onion was evaluated in this study.


DNA isolation from Aspergillus section Nigri isolates
Aspergillus section Nigri isolates were cultured on PDA medium, for two days at 28 °C.DNA was extracted using the cetyl trimethyl ammonium bromide buffer (CTAB) procedure from harvested growing mycelium as described by Ferracin et al. 15 .

PCR amplification, sequencing, and phylogenetic analysis
E1m4 (5P-TGR GGW GCW ACW GTT ATT ACTA-3P) and rE2m4 (5P-GGW ATA GMWSKTAA WAY AGC ATA -3P) were the forward and reverse primers, used in the amplification of cytochrome b gene based on the amino acid sequence, the primers were previously designed from cytochrome b amino acids 16 .The PCR reaction was done in a tube containing a total volume of 20 µL: 10 µL of 2X Tag master (Jena Bioscience, Germany), 0.5 µL from each primer (20 pmol/µL), 1 µL of the strain DNA (100 ng/µL), and 8 µL PCR ddH 2 O.The PCR reaction mixture was run for one cycle at 94 °C for 5 min, followed by 30 cycles consisting of denaturation for 1 min at 94 °C, annealing for 1 min at 50 °C, and extension for 2 min at 72 °C and final extension at 72 °C for 10 min.
The quality of obtained PCR products was checked on agarose gels (1%) stained with ethidium bromide and photographed by UV light on gel documentation (USA).
Sequencing and sequence analysis of the cytochrome b amplicons were done, the PCR products were purified using a Qiagene PCR purification kit (Qiagene, USA) and submitted to be sequenced (in Macrogen company, Korea).
Chromas Lite software was used to edit the cytochrome b gene sequences, and Clustal W software was performed to align them.Using MEGA software 6.0, the neighbor-joining tree with bootstrap values was created 17 .

Determination of ochratoxins A production by A. niger strains
According to Gabal et al. 18 , Aspergillus has an estimated ochratoxins potential.A conical flask containing 100 ml of YES broth with ingredients (g/L): sucrose 40 g and yeast extract 20 g was used to transfer an active culturing disc from a 7-day culture.After a two-week incubation period at 28 °C, the fungal filtrates were separated by filtration and then again filtered through glass fibre paper.The quantities of OTA were calculated in 10 ml that passed through an ochratoxins column (VICAM, Watertown, MA, USA) at 1-2 drops/s.The ochratoxins were eluted using 1.5 mL of ochratoxin elution solution after the columns had been washed twice with 10 mL of deionized water.The concentration of ochratoxins A was determined on a VICAM series-4 Fluorometer that had been recalibrated 19 .
Table 1.Macro and microscopic criteria of Aspergillus section Nigri on MEA medium at 28 °C for 7 days.

Characterization of Cement bypass dust and Zeolite
Cement bypass dust is environmentally harmful; therefore, getting rid of it and reusing it is preferable.The raw bypass dust was collected from a cement factory in Qift City (Qena governorate, Egypt).The Cement bypass waste was used without any modification or purification.The Cement bypass dust is characterized by a high concentration of alkalis, especially chlorides, K 2 O and SO 3 21 .El-Nahas et al. 22 described the method used to synthesize the Zeolite sample.Aluminium wastes were converted into Zeolite during the procedure using a household microwave.Finally, the collected Zeolite materials were filtered, rinsed with deionized water, and dried at 110 °C overnight.Zeolites are aluminosilicate solids with a negatively charged honeycomb structure of micropores that allow molecules to be adsorbed to catalyze Figure 1.Microscopic examination of isolated Aspergillus section Nigri, obtained from rotten onion bulbs, collected from Qena Governorate, Egypt, on MEA medium at 28 °C for 7 days.From left to right: 1-9: radiate and loosely radiate, uni and biseriate conidial head with ×40, 10-13: Conidia with ×100.chemical processes and clean up the environment.They are essential to green chemistry because they reduce the need for organic solvents 23 .

Fungal growth inhibition assay
Using a modified method of the fungal growth inhibition experiment reported by Fiori et al. 24 , the antifungal activity of Cement and Zeolite against A. niger was evaluated.The necessary concentration (as we tested different concentrations) 10 × 10 3 µg/mL was achieved by combining the powdered Cement or Zeolite with the melted PDA, this test was done by duplicate plates and the mean of them was calculated.The fungal isolate was cultured on PDA medium for 7 days, and an 8 mm-diameter disc of the growth was placed on the surface of the treated medium, incubation was done at 28 °C for 7 days.PDA medium without treatment was utilized for the control.After one week, the colony diameter was measured, and the percentage of fungal growth that was inhibited in comparison to the control treatment was determined using the following formula: where I is the percentage inhibition, C is the radial growth in control, T is the radial growth in treatment (test).

Management post-harvest bulb rots of onion
Cement and Zeolite powder were used to control the post-harvest healthy onions.The bulbs were cleaned and sterilized by ethyl alcohol (70%) for 1 min and dried by tissue under sterilized conditions.Thirteen bulbs for each treatment were covered by the tested powder, and sprayed by a concentrated spore suspension (1 ×10 8 spores/mL) of the tested fungal isolates.Thirteen untreated onion bulbs served as controls, sprayed by the fungal suspension.Bulbs without treatment and fungal suspension were also used.Onion bulbs were incubated at 28 °C for eight days.The percentage of rotten fruits and their disease severity were determined.Pathogenicity rating was recorded as the mean of duplicate bulbs for each isolate of A. niger, Less virulent: < 50%; Moderate: 50-75%; Highly virulent: more than 75% 25 .

Permission
All collected samples were transmitted to our lab in South Valley University after were complied with relevant institutional, national, and international guidelines and legislation and transferred safely to mycological analysis.

Mycobiota of stored bulbs
All the collected samples (n = 13) of stored bulbs were positive for fungal contamination with a percentage of 100% of Aspergillus section Nigri.Thirteen isolates of them were chosen for further characterization.

Macroscopic and microscopic examinations of Aspergillus section Nigri
Based on macroscopic and microscopic criteria, we identified the thirteen collected isolates as Aspergillus section Nigri in all onion samples.In all isolated strains, Aspergillus section Nigri strains were characterized macroscopically as a black colony with a narrow white border.Colony size was 29-50 mm.The colony texture was floccose, with white mycelium sparse on the colony.Microscopically: vesicle size was 33.958-82.736µm, vesicle shape was sub-globose to globose, conidial head was radiate and loosely radiate; uni and biseriate, conidia shape and color were sub-globose to globose; smooth or finely-to-distinctly roughened; colorless to brownish color.Conidia size was 2.459-7.862µm (Table 1 and Figs. 1, 2).

Molecular identification of Aspergillus section Nigri
Using the designed primer E1m-E2, the 437 bp fragments of the cytochrome b gene were amplified from all thirteen strains of Aspergillus section Nigri.Our strains of Aspergillus section Nigri showed similarity with 99-100% to the species of A. niger deposited at the Genbank.Based on the phylogenetic tree, the strains of Aspergillus niger were grouped with other A. niger sequences LC375152.1,AB000578.1,DQ178141.1,MT647904.1,achieved from the GenBank database with a bootstrap more than 80% (Fig. 3).The accession numbers of these isolates were inserted in the phylogeny tree.

Detection of ochratoxins A efficiency and pks gene of Aspergillus niger
Thirteen isolates of A. niger were subjected to ochratoxins A analysis.Three isolates were detected to produce ochratoxins A, and their production ranged from 1.5 to 15 ppm.Isolate of AEM7 was the highest producer of www.nature.com/scientificreports/ochratoxins A. Pks gene was not detected in all tested isolates, although three isolates exhibited the production of ochratoxins A (Table 2).

Characterizations of the synthetic Cement bypass dust and Zeolite
To determine the basic components presented in the disposal of Cement bypass dust and Zeolite, the X-ray Powder Diffraction Analysis (XRD) was studied at a 2θ range between 10° and 70°.As shown in Fig. 4, the results showed that several compounds were present, with calcite CaCO 3 being the most common component percentage in bypass dust (39.1%), followed by Larnite Ca 2 SiO 4 (25.2%) and Al 2 O 3 (14.4%).
Zeolite A showed the characteristic peaks at 2θ values: According to the Scherrer equation, the produced Zeolite was in the nanoscale range reached 21.7 nm, and had a cubic crystal structure with strong XRD peaks.
EDX (Energy Dispersive X-ray Spectroscopy) analysis provides the elemental composition of various constituent elements in the Cement waste materials.Ca contributed to 39.5% of the elemental composition.Si, Al, Na, Fe, and Cl had low percentages.
EDX analysis confirmed the elemental composition and constituent of tested types of Zeolite.Na, Al, Si, and O elements were observed in the tested sample.That confirms the purity composite phase with no impurities in Zeolite samples Fig. 5.

Antifungal activity of safely waste Cement and Zeolite
The two wastes inhibited the growth of collected isolates with different ranges.Waste of Cement exhibited maximum percentage growth inhibition more than Zeolite against the tested isolates.Waste of Cement showed the highest percentage of growth inhibition against AEM5 isolate at 86.7% and the lowest percentage of growth inhibition against AEM1 at 74.7%.In the case of Zeolite, the highest percentage of growth inhibition was observed in isolate AEM4 at 85.3%, and the low percentage was recorded in isolates AEM1 and AEM 10 at 49.3% (Fig. 6 and Table 3).

Protective effects of Cement and Zeolite on post-harvest bulbs rot disease of onion
The results revealed non-growth or slight to moderate growth of A. niger in treated bulbs.The two treatments significantly reduced bulbs rot disease of onion than untreated bulbs.Seven strains of A. niger were completely suppressed by Cement treatment, and they were AEM3, AEM5, AEM6, AEM8, AEM 10, AEM12, AEM14.Nine isolates were inhibited in the growth when covered with Zeolite treatment; they were AEM3, AEM4, AEM5, AEM6, AEM8, AEM9, AEM12, AEM13, AEM14 (Figs. 7 and 8).

Discussion
Thirteen isolates of Aspergillus section Nigri were isolated from the samples (n = 13) of stored onions from markets in Qena.All the tested onions showed contamination by black Aspergilli; which were characterized by morphological, physiological and molecular criteria.Under hot and humid conditions, black mold (A.niger) is a serious contaminant of onion bulbs 5,26 .Earlier mycological studies confirmed the fungus's ability to spread via seeds, soil, and the air 27 .Aspergillus niger was demonstrated to be transmitted from them to developing seedlings and onion sets 28 .When the aerial leaves die, the fungus either enters through the neck, wounded roots, or scales 29 .However, harvesting onions before the leaves are fully dried results in more storage rots and lowers the market value of the bulbs 30 .
Varga et al. 31 examined six samples of onion for the presence of black aspergilli.They were able to effectively isolate black aspergilli from each sample examined.According to Gherbawy et al. 32 , 37 onion samples from the Taif region contained black aspergilli.Aspergillus section Nigri was expected to be isolated from 98 percent of the onion samples.Multiplex PCR allowed for the differentiation of A. niger/A.welwitschiae strains from other Aspergillus section Nigri species.According to Massi et al. 6 , A. niger/A.welwitschiae was significantly more prevalent than other A. niger aggregate species among 500 randomly selected fungal isolates.
The Aspergillus niger "aggregate" consists of a variety of morphologically similar species of Aspergillus section Nigri, including A. niger, A. brasiliensis, A. tubingensis, A. welwitschiae, and A. luchuensis 8 .Morphological and molecular identification were used to confirm the characterization of black Aspergilli in this study.The analysis of the phylogeny, classification, and identification of black Aspergilli can be done using the cytochrome b sequences.The first study using the mt cytochrome b gene in the phylogenetic analysis of fungi was Wang et al. 16 .Due to www.nature.com/scientificreports/ the discovery of species-specific sequences in the cytochrome b gene, Aspergillus strains can be distinguished according to the species level.For Aspergillus strains, conventional morphological identification is challenging or unavailable, examining the sequences is a helpful tool for precise identification and taxonomy.Information about evolutionary relationships among interspecies to intraspecies level can be found using the cytochrome b gene.This study found that three isolates out of thirteen exhibited ochratoxins A production, and none of them showed the tested PKS gene.Genes involved in mycotoxin production are frequently grouped, co-regulated, and co-expressed in fungal genomes.Genes encoding one or more types of "core" enzymes required for producing the metabolite backbone structure are typically found in these gene clusters 33 .They may contain genes for regulatory proteins, hydrolases, oxidases, methylases, and other enzymes that are involved in adjusting the backbone structure to make the final metabolite, such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) genes 34 .
Ferracin et al. 15 examined 119 A. niger strains for the presence or absence of the pks gene as well as their ability to produce OTA.The presence of the pks locus in the A. niger/A.welwitschiae genome was positively correlated with the capacity to produce OTA, as demonstrated by the detection of the pks gene in every strain that produced OTA (n = 31) but not in any non-producing strains (n = 88).Our investigation did not find the pks gene in all strains tested, even though three of the examined isolates were positive producers.This may have been caused by a lack of amplification product or a low pks gene concentration or the production was correlated to pks gene region not studied here.A. carbonarius has demonstrated the ability to be toxic, as demonstrated by the culture plug method 35 , but its toxigenic (pks) gene was not amplified.Numerous investigations have demonstrated that the polyketide synthase (pks) and nonribosomal peptide synthetase (NRPS) genes influence the OTA production pathways in Penicillium and Aspergillus species 36 .A class of multifunctional proteins that includes both PKSs and NRPSs is involved in the synthesis of numerous secondary metabolites with a broad variety of biological functions.Amino acids and carboxylic acid moieties are the starting points for the biosynthesis of a large range of naturally occurring microbiological products.These hybrid NRPS-PKS systems, which involve direct interactions between NRPS and PKS modules, may accelerate the production of these mixed peptide-polyketide natural products, or they may work separately to build the peptide-polyketide backbone 37 .
This study characterization of Cement waste and Zeolite agreed with Salem et al. 38 .The presence of calcium hydroxide in CEM Cement is responsible for its effective antifungal activity, as shown by the research of Ayatollahi et al. 39 , who found that it inhibited the growth of Candida albicans except in the first few days.When CEM Cement's calcium hydroxide comes into contact with a medium, it breaks down into Ca ++ and OH, which raises the pH.Inhibition of cell membrane enzymes occurs at alkalinities above nine 40 , meaning that such conditions can inhibit cellular activity.
When tested against Candida albicans, it was shown that Zeolite-Cu 2+ and Zeolite-Zn 2+ displayed a higher zone of inhibition.According to Cardoso et al. 41 , Zeolites are nanoporous alumina silicates in a framework with cations that display ion-exchange characteristics with metal ions, making them potential antimicrobial materials.According to Ozogul et al. 42 , Zeolite can be used as a natural addition to stop various biogenic amines, like cadaverine (CAD) and putrescine (PUT), from being formed by Gram-negative bacteria.
In conclusion, storing the bulbs in clean stores and covering them with Cement and Zeolite is an eco-friendly and safe alternative to synthetic fungicides against post-harvest rot of onion bulbs, it will most probably prolong the storage life of the bulbs against Aspergillus section Nigri that causes post-harvest rot.

Figure 2 .
Figure 2. Colony colour, and texture of tested Aspergillus section Nigri, isolated from onion bulbs on MEA medium at 28 °C for 7 days.

Figure 3 .
Figure 3. Phylogenetic tree reconstructed from the cytochrome b gene sequences of isolated Aspergillus niger aligned with the other sequences from Aspergillus section Nigri deposited in the NCBI database.Candida albicans was used as the outgroup.The numbers above branches indicate bootstrap values that were constructed after a run of 1000 replications.

Figure 4 .
Figure 4. XRD pattern of the Cement waste and Zeolite.

Table 2 .
Codes, accession numbers, ochratoxins A exhibited by A. niger isolates, and surveying of PKS gene.

Table 3 .
Effect of Cement and Zeolite on the fungal growth causing post-harvest rot of onion.Figure 7. Percentage of rot after inoculation of onion bulbs with isolated Aspergillus niger isolates, after storage period at 28 °C for 7 days.